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BluePippin User Manual

1. cccceeeseeeeeeeeeeneeeeeeeeeeeeeeeeeeneeeenens 6 6 6 6 Assigning Reference Markers internal standardS siria 6 7 6 7 Warnings and INGICAlOrS iui 6 8 PROGRAMMING SIZE SELECTIONS TIGHT RANGE AND TIMED MODES 7 1 7 1 Tight Mode programming minimum size range collections 7 1 7 2 Range Mode programming broad size range collections 7 3 7 3 Time Mode programming timed collections rrrrrrreiiiiiieiieiiiiiienn 7 4 PROGRAMMING DNA BAND CAPTURE WITH MIDORI GREEN PEAK MODE 8 1 OPTICAL CALIBRATION ae ns saines 9 1 PREPARING A CASSETTE 2 sree se terse a aN E aE 10 1 10 1 Visually Inspect the Cassette 1s ssrrrrrrrrreee iii iii 10 1 10 2 Prepare the Cassette for Loading ss mnsssssnnnrrreennsnnnnereeennnnnnnnnnnne 10 3 103 CORAN SR a EEEE aE 10 4 10 4 Adding Midori Green for Band Capture Cassettes 1111srrrrrsssssezezzono 10 6 LOADING SAMPLES criari ranieri anta 11 1 BluePippin Operations Manual software v 6 13 cassette definition Set 15 12 13 14 15 16 17 18 19 20 21 22 23 24 BluePippin Operations Manual software v 6 13 cassette definition Set 15 888 744 2244 www sagescience com support sagescience com RUNNING A PROT OGG 5 snmmnstsammsssmmmonsnotcmnnsm me annsms nes cnsmmesmssenses 12 1 PAT OVON OW acilia 12 1 122 AIO RO nn 12 1 12 3 Mo niornng a RUN acei
2. Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 8 888 744 2244 www sagescience com support sagescience com 24 8 0 75 Agarose Dye Free 1 6 kb Marker S1 Low Voltage external marker Use Product Number BLF7510 Use Cassette Definition 0 75 Agarose Dye Free 0 75 DF Low Voltage 1 6kb Marker S1 DNA Marker S1 Typical times to detector l l l 7 02 00 0300 O4 00 05 00 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets bp hr min 1 000 3 04 Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 9 888 744 2244 www sagescience com support sagescience com 24 9 0 75 Agarose Dye Free 1 6 kb Marker S1 Pulsed Field external marker Use Product Number BLF7510 Use Cassette Definition 0 75 Agarose Dye Free 0 75 DF 1 6kb Marker S1 DNA Marker S1 Typical times to detector l ss 1 01 00 01 30 0200 02 30 Time hmm Estimated Collection Times for Minimum Size Range Tight Targets bp hr min Run times
3. sage science Blue Pippin DNA Size Selection System Operations Manual Software v 6 13 Cassette Definition Set 15 888 744 2244 www sagescience com support sagescience com Sage Science Inc Suite 2400 500 Cummings Center Beverly MA 01915 2014 Sage Science Inc All rights reserved Sage Science and BluePippin are trademarks of Sage Science Inc All other brands and name mentioned herein are property of their owners BluePippin Operations Manual software v 6 13 cassette definition Set 15 I ER N 10 11 888 744 2244 www sagescience com support sagescience com TABLE OF CONTENTS INTRODUCTION rane 1 1 SAFEFGCANDRREGALTFIONS acirinioniaiinezinzisonsialenazinziotaaziniananina fata zana eten 2 1 UNPACKING AND INSTALLATION rr 3 1 ACHIEVING BEST RESULTS FROM THE BLUEPIPPIN cccssseccteseeeeseseeseeneeeseens 4 1 4 1 Keys to Successful Collections cccccsssessseeeseeenseeeeeeeensseeseeoenseeeseennseeessoeesseeeseoeenseeesons 4 1 4 2 COMMON MISCONCEPTIONS ccceceeeeeeeeeeeneeeeeeeeeceeenneeeeeeeeeoeessseeeeeeeeeoonasseseeeeeeeooensneeeeees 4 1 SAMPLE PR PARATION reed 5 1 PROTOCOL PROGRAMMING GUIDE nti 6 1 OM OVEIVICW PR ES 6 1 6 2 ProgrammingSsumMavd ia 6 2 6 3 Selecting a Cassette Definition rien 6 4 6 4 RUN LIMES car E 6 5 6 5 Assigning a Reference Marker external Marker
4. Sample Well Elution Well Buffer Chambers Buffer Chamber Optical Detection sufi Region Figure 10 3 The region of optical detection BluePippin Operations Manual software v 6 13 cassette definition Set 15 10 2 888 744 2244 www sagescience com support sagescience com 10 2 Prepare the Cassette for Loading 1 Dislodge bubbles from behind the elution wells Return the cassette to the right side up position Tilt the cassette sample well side down to release any trapped bubbles behind the elution modules into the buffer chambers Gently tap the cassette if necessary Figure 10 4 below shows location to check for bubbles Sample Well Elution Module Buffer Buffer Chamber 1 gt TT tes Chambers ____t L iO D ad 7 T _ nl ____ G2 LA GJ x bar Ti Il o i T lt 9 i RI lf Check behind elution modules for bubbles Tilt cassette to clear ri i 2 eg i Figure 10 4 A schematic of a Gel Cassette 2 Place Cassette into the Pippin optical nest The cassette should be placed into the nest in the orientation shown in Figure 10yi 4 above with the sample wells to the left side of the nest When inserting the cassette into the nest keep the buffer chambers tilted down so that the bubbles in the elution reservoirs won t be trapped behind the elution modules Important Be sure the cassette is ful
5. Peak Mode Band Capture cassettes are used to collect DNA bands such as PCR or restriction fragments These cassette require the addition of an DNA binding dye Midori Green to the buffer chambers of the separation channels of the sample lanes see Section 9 4 Band capture uses the Peak mode for programming Using Peak mode a base pair value is entered into a threshold field The BluePippin will collect the next band that the system detects after the programmed base pair threshold The amount of DNA loaded should be between 50 ng 2 ug band Larger band gt 0 5 ug require additional programming modification to account for band broadening Peak mode requires the use of an external marker for reference In the protocol editor 1 Select an appropriate cassette type for size selection if a new protocol is being created 2 Assign external reference DNA marker a enter the lane into which the marker will be loaded into the Reference Lane field 1 is the default value b press the APPLY REFERENCE TO ALL LANES button 3 Select the Peak programming mode Click the Peak button for the lane from which the fragment peak will be extracted 4 Enter a value in for the BP Threshold Enter a base pair value that precedes the beginning of peak band of the fragment to be collected If possible use a threshold value that is lt 80 90 of the beginning of the leading edge of the t
6. The specified starting volume of 40ul only partially fills the module 5 Seal the elution wells with the adhesive tape strips Tape for sealing the elution wells are supplied with cassette packaging Long elutions will cause the elution wells to overflow if not sealed Place tape over the elution wells and rub firmly to fix the tape in position For best results rub the tape at the boundary of the well with a smooth hard object such as the back end or a lab marker pen 6 Check the buffer levels in the sample wells Sample wells should be completely filled to the top with buffer If any wells are under filled top them off with additional buffer Note The total volume of the sample well is approximately 70 ul 10 3 Continuity Test The continuity test measures the current in each separation and elution channel and determines whether they are within the expected values for a successful run Important Temperature affects electrical current readings If cassettes have been refrigerated they will fail the current test until the cassette is at least 17 C 62 F Should this be the case wait until the cassette temperature has equilibrated to room temperature and re test 1 Press Test With the cassette in the optical nest close the lid and press TEST on the controller on the Main Tab TEST START CALIBRATE SNAPSHOT INFO SHIITE ON BluePippin Operations Manual software v 6 13 cassette definition Set 15 10 4 888
7. 44 2244 www sagescience com support sagescience com 2 Continuity Test will Automatically Run The continuity test screen will launch The separation and elution channel test parameters are displayed and the electrical current results will be listed after several seconds If the test returns a PASS message press RETURN and continue to baseline calibration Continuity Test MEN DELE REED EEN 2 EE MEXTEE i Failed Continuity Tests 1 A failed test is indicated by a FAIL message and the failed channel is highlighted in orange Figure 10 5 shows a failed test screen elution channel in lane 1 2 If a Separation lane has failed left column continuity Do not use that lane Remaining passing lanes can be used if necessary 3 If an Elution channel right column has failed continuity Replace buffer in the elution module of the failed lane and refill with 40 ul of fresh electrophoresis buffer and retest the cassette If the lane fails again do not use the lane for collections Continuity Test separate max mA mA Test parameters separate min mA elute min mA 1 Elution Channel Separation Channels RELA ution Channels FAN edono eo 0 i FAIL replace buffer and retest If it fails again do not use for collections RETURN BluePippin Operations Manual software v 6 13 cassette definition Set 15 10 5 888 744 2244 www sagescience com support sagescience com Figure 10
8. Note f there is a time value in the Elution Timer field and the Separate indicator is green then the elution has been completed BluePippin Operations Manual software v 6 13 cassette definition Set 15 12 4 888 44 2244 www sagescience com support sagescience com The Elution Timer records the length of time of the elution When elution is complete electrophoretic path will switch back to the separation channel and the elution timer will show the value of the total elapsed elution time Elution Timer Reference Idle Separate Elute Run progress is monitored in the Protocol Status panel The percent complete is indicated by a progress bar The Clock fields displays the current time and date The Estimated Completion Time displays the estimated time of protocol completion This value is updated and more accurate after DNA markers have been detected The Remote Access indicator is activated if the instrument is being remotely controlled from a PC via VNC server Contact Sage Science for instructions on remote control Protocol A 24 Feb 2012 05 41 52 24 Feb 2012 05 41 45 BluePippin Operations Manual software v 6 13 cassette definition Set 15 12 5 888 744 2244 www sagescience com support sagescience com 12 4 Log Files At the end of every run the BluePippin will automatically save a log file txt which can be viewed on the Log Review Tab or the PC software version of Log Review A
9. Samples Proper sample loading is critical for best performance of BluePippin cassettes For maximum reproducibility and accuracy the sample should travel through the central section of the gel column and should be bounded on all four sides by uniformly conductive media either gel or electrophoresis buffer The goal of the loading procedure is to produce this geometry in the sample loading well as illustrated in the bottom section of Figure 11 1 Properly prepared samples will be 40 ul in total volume consisting of 30 ul of DNA mixed with 10 ul of BluePippin loading solution The loading solution contains concentrated Ficoll as a densifying agent see following chapter on Sample Preparation for details and therefore the samples will sink and form a high density layer beneath the electrophoresis buffer when pipetted slowly into the sample wells If there is insufficient conductive buffer over the sample the electrophoretic forces lines will curve upward as the sample exits the well see Figure 11 1 upper section and the sample will be drawn to the top of the cassette where it can travel out of the gel into the gap between the gel column and the plastic top of the channel Sample moving in this gap will travel at a different rate than the sample inside the gel column and will lead to elution of undesired size fractions in the eluted material In such cases the contaminating DNA will usually but not always be higher in molecular weight than th
10. Start BP End Start Exp N End Exp 0 90 b 1 10 90 of BP Start value 110 of BP Start value Figure 8 1 Expansion factors values widen the peak collection timing to ensure complete capture of the peak BluePippin Operations Manual software v 6 13 cassette definition Set 15 8 2 888 744 2244 www sagescience com support sagescience com Expansion factors for larger sample loads For sample loads of 0 5 ug band and below the default expansion factors should result in successful collection of the entire DNA band Higher sample loads will migrate slower relative to the reference markers and exhibit band broadening these instances users should increase the End Exp values to capture the full amount of DNA The Start Exp should remain at 0 90 This is illustrated in Figure 8 2 The maximum End Exp value that might be required is 1 50 BP Start BP End Start Exp AN End Exp 9o00 nf RD Pie 2 A NO F DA Cazzi va YUYo O BI Start value 10 OT BP Start value Figure 8 2 Default values for sample loads of 5 ug and below should be sufficient for most collections Larger sample loads will require higher End Exp values to collect the entire band Start Exp values needn t be adjusted for larger loads 6 Enter Sample ID information 7 Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new p
11. The Current graph allows users to closely inspect the electrophoretic current profile of a Pippin run from a run log The colored horizontal traces display the actual current signal results in mA The timing of an elution switch from separation to elution is accompanied by a 0 5 mA drop in current ay CURRENT EL 4 50 4 00 3 50 3 00 2 50 2 00 Lane Current mA 1 90 1 00 0 50 0 00 l l l l 01 05 48 01 08 00 01 10 00 01 13 02 Time hh mm ss There are three icons in the upper right hand corner of the graph The functions are listed below Revert to default Grab and move view screen Zoom hold left click for Options Saving a Review Image An image from the review screen may be captured and automatically saved by pressing the Snapshot button The png file will date and time stamped and saved in the Pippin log file directory SNAPSHOT BluePippin Operatid VidliUudl SUILWale V O CASSEL UCI Oli Det 15 18 5 888 744 2244 www sagescience com support sagescience com 19 Managing Files File Manager Tab The default screen on the BluePippin is a tabbed format The File Manager Tab is the fourth tab 18 1 Overview The File Manager Tab allows users to access copy and delete BluePippin files There are three types of files used by the system log text and image protocol and cassette type The screen is divided into two sub screens the Pippin file directory on th
12. all sample collections will not include the field reversal The default setting is off When activated the current will be reversed on all subsequent runs until the user changes the setting through the System Options tab Date Time This allows users to set or change the date and time settings for the instrument The date and time settings are recorded in log files and log file names BluePippin Operations Manual software v 6 13 cassette definition Set 15 16 1 888 744 2244 www sagescience com support sagescience com To Change the settings press the System Options Tab Instrument name 1 Enter a text into the Instrument Name field 2 The Settings Changed indicator light will activate Press Accept to accept the new setting Reverse field option 1 Toturnon press the Use Reverse Field for Sample Recovery button The indicator display will change from dark gray to white and the display will change from OFF to ON 2 The Settings Changed indicator light will activate Press Accept to accept the new setting A Important The reverse field setting will apply to all runs until it is changed in this tab Date and time 1 Edit the date or time display directly in the Time Disipay field or select the calendar icon next to the field and edit the time or select a date 2 The Settings Changed indicator light will activate Press Accept to accept the new
13. screen image png of the Main Tab is saved after the run has been finished Screen images of all continuity tests and calibration tests are also automatically saved All files may be accessed from the File Manager Tab The files are saved internally on the Pippin hard drive in the directory named home pippin BluePippin Logs in a folder with a year month YYYY MM folder name Log files names have the following convention software version year month day hour minute second _ user input protocol name or test type file extention An example of the four types of log files and file structure in the File Manager Tab is shown below The files will be saved every time the these procedures are undertaken Main Protocol Editor Log Review File Manager Pippin Folder p home BluePippin Logs 2012 02 Pippin Listing fone level upi P5 2012 02 15 11 28 22 LEDCalibration png Le PS 2012 02 15 11 28 22 ProtocolA txt P5 2012 02 15 11 28 22 ProtocolA png PS 2012 02 15 11 05 39 Continuity png BluePippin Operations Manual software v 6 13 cassette definition Set 15 12 6 888 744 2244 www sagescience com support sagescience com 13 Sample Collection Sample Well Elution Module _ ale buffer Buffer Chamber A I gt Chambers i DE f 2 a D a F i x te r Pi gt ee a Le i rn N i ra gt J r et i Ps f 1 a e Lili 13 1 Overview Rem
14. setting Instrument Name Blue 2 UDP Server Port Use Reverse Field for Sample Recovery Date Time A change in settings will not be accepted if an experiment is underway REVERT Settings Changed ACCEPT BluePippin Operations Manual software v 6 13 cassette definition Set 15 16 2 1 888 44 2244 www sagescience com support sagescience com Running in Manual Mode The BluePippin may be run without using a programmed protocol and with manual control 1 Select the Manual Mode from the Protocol Name drop down menu in the Run File Manager Manual Mode 07 Feb 2012 17 23 08 07 Feb 2012 20 23 07 2 Press Start on the controller 3 Sample lanes may be run individually by pressing the Separate button and elutions are carried out by pressing the Elute button After elution press the Separate button to end the elution and return to separate mode or press the Idle button to stop the run on that lane Sample ID Current Elution Timer Reference Idle Separate Elute BluePippin Operations Manual software v 6 13 cassette definition Set 15 17 1 888 744 2244 www sagescience com support sagescience com 4 Press Pause Resume and Stop buttons are also operative as in automated runs TESI START CALIBRATE SNAPSHOT INFO SHITE Note There is a delay between the time that a fragment is detected and visible on the graph or O image and
15. that require a long elution period The T pause value must be within the extraction range between T Start and T End The pause function temporarily turns off power to the electrodes and suspends run timers allowing users to remove the sample rinse the elution well if desired and then resume the instrument run to collect additional sample A Important A pause will require the user to manually resume the protocol from the Main Screen controller The instrument lid may be opened during the pause and sample retrieved BluePippin Operations Manual software v 6 13 cassette definition Set 15 7 4 888 744 2244 www sagescience com support sagescience com 4 Enter Sample ID information 5 Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new press Save If a protocol has been edited a yellow alert will be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As will allow a new name for the file to be applied Protocol Changes NOT Applied SAVE AS All protocol files are saved in a directory home pippin BluePippin Protocols and may be accessed in the File Manager tab BluePippin Operations Manual software v 6 13 cassette definition Set 15 7 5 888 744 2244 www sagescience com support sagescience com 8 Programming DNA Band Capture with Midori Green
16. 102 Filtered Ref Threshold BluePippin Operations Manual software v 6 13 cassette definition Set 15 18 3 888 744 2244 www sagescience com support sagescience com Signal Graph The Signal graph allows users to closely inspect the elution results of a Pippin run from a run log The colored horizontal traces display the actual optical signal results in mA The vertical red lines indicate called reference DNA marker peaks that are listed in the DNA marker times and values list There is an Analyze Peaks button to recall the markers The yellow vertical line indicates the estimated base pair position at a given run time The indicator may be moved see below along the x axis showing the relative base pair position If the perspective of the graph is changed see below the indicator will automatically default to the center of the x axis SIGNAL El bl 0 82 0 70 0 60 I 050 T 5 5 040 Ci PL CL 0 08 l I I I I l l Il 01 05 48 01 07 00 01 08 00 01 04 00 01 10 00 011100 011200 01 13 02 L Time hh mm ss There are four icons in the upper right hand corner of the graph The functions are listed below Revert to default view Grab and move screen Move yellow Zoom base pair hold left indicator click for Options BluePippin Operations Manual software v 6 13 cassette definition Set 15 18 4 888 744 2244 www sagescience com support sagescience com Current Graph
17. 4 www sagescience com support sagescience com 24 13 0 75 Agarose Dye Free 18 27 kb Marker T1 Pulsed Field external marker Use Product Number BMF7510 Use Cassette Definition 0 75 Agarose Dye Free 0 75 DF 18 27kb Marker T1 DNA Marker T1 Typical times to detector l l l 02 00 02 30 03 00 TERJI Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets Target Time to Time to Collect bp hr min 18 000 3 49 23 000 4 00 27 000 4 15 Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Accuracy lt 10 Reproducibility lt 10 Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 14 888 744 2244 www sagescience com support sagescience com 24 14 0 75 Agarose 50kb Targets Marker Z1 Pulsed Field external marker 50kb Use Product Number BHZ7510 Use Cassette Definitions 0 75 Agarose Dye Free 0 75 DF 50 kb Marker Z1 tight 0 75 Agarose Dye Free 0 75 DF 50 kb Marker Z1 broad DNA Marker Z1 Typical times to detector Important Range mode programming is not allowed for this product Only Target mode can be used Two definitions are 4 00 05000 0600 07 00 provided Tight pr
18. 5 The Continuity Test screen The elution channel in lane 1 has failed 10 4 Adding Midori Green for Band Capture Cassettes Red Separation Channel Buffer Chambers Sample Well Elution Module a add Midori Green Dye Buffer Chambers Buffer Chambers Pal Pa Pa x r Optical Check for bubbles behind Region elution modules Tilt up to clear Figure 10 6 Cassette schematic illustrating to which buffer chambers Midori Green must be added shown in red For Cassettes that require Midori Green additional pippeting and mixing steps are required Midori Green is a DNA binding dye which must be added to the separation channel buffer chambers When a run is intiated the Midori Green dye will migrate in the reverse direction to the DNA bind to it and allow visualization by the BluePippin optics To add Midori Green to a cassette 1 Remove Midori Green dye provided with cassette kits from refrigerator Briefly vortex and spin 2 Pipette 40ml of Midori Green dye into each of the five separation channel buffer chambers show in red above Take care not to pippette into the elution channel chambers 3 Using a P1000 set to 750ml mix the dye by slowly aspirating and dispensing the chamber contents in the five separation chambers BluePippin Operations Manual software v 6 13 cassette definition Set 15 10 6 11 888 744 2244 www sagescience com support sagescience com Loading
19. 75 DF Marker S1 High Pass 15 20kb DNA Marker S1 Typical times to detector 5 RU ne i i l 00 0 00 45 01 00 01 15 Time hEmm Estimated Collection Times Approximately 2 hours for all BPStart settings Performance Specifications Accuracy and reproducibility are affected by the sample load amount Refer to the High Pass user guide for recommendations Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 12 888 744 2244 www sagescience com support sagescience com 24 12 0 75 Agarose Dye Free 10 18 kb Marker U1 Pulsed Field external marker Use Product Number BUF7510 Use Cassette Definition 0 75 Agarose Dye Free 0 75 DF 10 18kb Marker U1 DNA Marker U1 Typical times to detector l l l 02 00 02 720 0240 0300 0320 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect bp hr min 10 000 2 55 12 000 3 00 15 000 3 15 17 000 3 25 18 000 3 35 Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 13 888 744 224
20. LL LANES button 3 Select the Tight programming mode Click the Tight button everly lane in which the Tight fragment range will be collected If using and external marker make sure no collection modes are selected for that lane 4 Enter a value in for the BP Target This value should represent the median value for the desired size range to be collected The actual range to be collected will auto fill the BP Start and BP End fields BluePippin Operations Manual software v 6 13 cassette definition Set 15 1 1 888 744 2244 www sagescience com support sagescience com 5 Optional Enter a BP pause value If a value is entered into the pause field the system will pause when it has estimated that that base pair value has been collected during the elution of that lane A pause will allow a user to remove two consecutive cuts from a sample The two cuts will usually have overlapping size distributions User may wish to use the pause feature for two reasons 1 to retrieve nearly identical cuts from the same sample or 2 to avoid overflow of the elution module during collections that require a long elution period The BP pause value must be within the extraction range between BP Start and BP End The pause function temporarily turns off power to the electrodes and suspends run timers allowing users to remove the sample rinse the elution well if desired and then resume the instrument run to collect addi
21. Marker Q2 internal standards 24 2 24 2 2 Agarose Dye Free 100 bp 600 bp Marker V1 internal standards 24 3 24 3 2 Agarose Dye Free 100 bp 600 bp Marker X1 int stds 25 ul elution well 24 4 24 4 2 Agarose Dye Free 100 bp 600 bp Marker M1 external marker 24 5 888 744 2244 www sagescience com support sagescience com 24 5 1 5 Agarose Dye Free 250 bp 1 5 kb Marker R2 internal standards 24 6 1 5 Agarose Band Capture 250 1500 bp Marker A1 external marker 24 7 24 7 0 75 Agarose Band Capture 2 10 kb Marker D1 Pulsed Field external marker 24 8 24 8 0 75 Agarose Dye Free 1 6 kb Marker S1 Low Voltage external marker 24 9 0 75 Agarose Dye Free 1 6 kb Marker S1 Pulsed Field external marker 24 10 0 75 Agarose Dye Free 3 10 kb Marker S1 Pulsed Field external marker 24 11 24 11 0 75 Agarose Dye Free High Pass Protocols external marker 24 12 24 12 0 75 Agarose Dye Free 10 18 kb Marker U1 Pulsed Field external marker 24 13 24 13 0 75 Agarose Dye Free 18 27 kb Marker T1 Pulsed Field external marker 24 14 24 14 0 75 Agarose 50kb Targets Marker Z1 Pulsed Field external marker 50kb 24 15 BluePippin Operations Manual software v 6 13 cassette definition Set 15 888 744 2244 www sagescience com support sagescience com Introduction Thank yo
22. agescience com support sagescience com Figure 3 4 BluePippin front panel USB Port Indicator Light Indicator Light Green when electrophoresis is Blue when instrument is underway protocol is running ready for use power is on and software is active BluePippin Operations Manual software v 6 13 cassette definition Set 15 3 4 888 744 2244 www sagescience com support sagescience com Unpacking Gel Cassettes Gel cassettes are shipped in boxes in the following configurations Ensure boxes are in the upright position and confirm that following contents are present Dye free gels with internal standards 50 samples size selections 90 bp 1 5 kb o 10 foil sealed gel cassettes store at R T o 1 package of adhesive tape for sealing elution wells o 1 reagent kit for 10 cassettes store at 4 C 550 ul DNA internal standards loading solution mix 10 ul sample 40mlofspare running buffer Dye free gels with labeled external marker 40 samples size selections gt 2kb o 10 foil sealed gel cassettes store at R T o 1 package of adhesive tape for sealing elution wells o 1 reagent kit for 10 cassettes store at 4 C 40 ml of spare running buffer 440 ul DNA size markers 1 x 40 ul load cassette 500 ul loading solution 10 ul sample 2X 800 ul 0 1 Tween20 in running buffer 40 ml of spare running buffer DNA binding dyes with external marker o 10 foil sealed gel casse
23. agescience com support sagescience com 10 Preparing a Cassette 10 1 Visually Inspect the Cassette 1 Remove the cassette from foil packaging Foil package is scored at one end to allow tearing 2 Inspectthe levels of buffer in the buffer reservoirs Tip the cassette to consolidate any bubbles in the reservoirs then hold the cassette in a horizontal position and look at the reservoirs from the top and bottom edges Reservoirs should be nearly full of buffer and roughly equal in volume across the cassette If the buffer level in any reservoir appears less than 50 full Compared with its neighbors the low reservoir should be refilled prior to running When refilling a visual check of buffer levels is sufficient a measured volume is not required side of cassette buffer volumes are Low side of cassette Figure 6 1 Low buffer levels in cassette buffer chambers 3 Inspect the gel columns Look for obvious breakage of the agarose column in each channel A Important f there is obvious breakage do not use the lane Remaining lanes can be used BluePippin Operations Manual software v 6 13 cassette definition Set 15 10 1 888 744 2244 www sagescience com support sagescience com 4 Inspect bottom of cassette for bubbles in the detection region of the gel columns If a cassette has been jarred during shipping the agarose column can delaminate from the plastic bottom of the separation channel forming a th
24. are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 10 888 744 2244 www sagescience com support sagescience com 24 10 0 75 Agarose Dye Free 3 10 kb Marker S1 Pulsed Field external marker Use Product Number BLF7510 Use Cassette Definition 0 75 Agarose Dye Free 0 75 DF 3 10kb Marker S1 DNA Marker S1 Typical times to detector I I l l l l 01 20 01 40 0200 02 20 02 40 0300 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets bp hr min Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications Minimum Size Distribution as CV Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 11 888 744 2244 www sagescience com support sagescience com 24 11 0 75 Agarose Dye Free High Pass Protocols external marker Use Product Number PAC20KB or BLF7510 Use Cassette Definition Version 2 0 75 DF Marker S1 high pass 4 10kb vs2 Version 3 0 75 DF Marker S1 high pass 6 10kb vs3 0
25. arget band The BluePippin will automatically collect the next peak that is detected The mass of minimum detectable band is approximately 50 ng Peak collection cannot be used with dye free cassettes Important t is important to verify the size of the band by electrophoresis Agilent Bioanalyzer M agarose gel BluePippin Operations Manual software v 6 13 cassette definition Set 15 8 1 888 744 2244 www sagescience com support sagescience com BP Thresh Start Exp End Exp Sample ID Template 9000 0 90 E 110 ______uyy 9000 D 090 E 110 _______ __ k 5 EE 000 D 0 20000 090 fino fl 5 Adjust Expansion factor values if needed This is recommended for loads above 0 5 ug band About Expansion factors When the optical detector identifies a band this occurs when post BP Thresh the signal increases over 10 above baseline the system will determine collection timing and estimate the peak size based on the reference marker calibration As is the case with size selections in tight mode the instrument will determine the base pair value at which to begin collection BP Start and at which to end collection BP End The expansion values with default values of 0 9 and 1 10 respectively automatically widen this range to ensure that the entire peak is collected This is illustrated in Figure 8 1 below Expansion factors values may be used to widen or narrow the collection criteria based on user needs BP
26. bp Conc ng ul 3 60 20 0 e Tide ay ee 300 400 500 00 1500 Figure 14 2 Example of a Bioanalyzer result for the 150 bp target of a Control DNA run on a 2 0 Agarose Cassette Important Data are not intended to imply guaranteed results or performance Control AM standards are intended to demonstrate that the BluePippin system is functioning as expected and that proper operational technique is being used BluePippin Operations Manual software v 6 13 cassette definition Set 15 15 2 888 744 2244 www sagescience com support sagescience com 16 System Options Tab The default screen on the BluePippin is a tabbed format The Protocol Editor is the fifth tab Overview The System Options Tab allows users to set or change global configurations for the software and BluePippin Instrument Name Reversible Field System Options Tab UDP Port SA Date and I 25 Feb 2012 020459 O Time e accepted if an experiment is underway ged ACCEPT This screen allows users to set the following Instrument name A name can be given to or changed for the instrument that will be displayed in the Main Tab and in the log files UDP Server Port Provides and address for remote communications between the BluePippin and wireless control Contact Sage Science for support Reversible Field for Sample Recovery When activated all sample collections will terminate with a 5 second field reversal to improve recovery When deactivated
27. cience com support sagescience com 24 6 1 5 Agarose Band Capture 250 1500 bp Marker A1 external marker Use Product Number BSG1510 Use Cassette Definitions 1 5 Band Capture 250 1500bp Marker A1 DNA Marker A1 Typical times to detector I I 00 30 00 40 00 50 01 00 01 10 01 20 Time hh mm Estimated Collection Times for DNA bands Time to Time to Collect ra Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 7 888 744 2244 www sagescience com support sagescience com 24 7 0 75 Agarose Band Capture 2 10 kb Marker D1 Pulsed Field external marker Use Product Number BLG7510 Use Cassette Definitions 0 75 Agarose Dye Free 0 75 Band Capture 2 5kb Marker D1 0 75 Agarose Dye Free 0 75 Band Capture 5 10kb Marker D1 DNA Marker D1 Typical times to detector 01 00 01 50 03 00 01 00 01 30 02 00 0230 Time hh mm Tive hhinn 2 5 kb protocol 5 10 kb protocol Estimated Collection Times for DNA bands Target Time to Time to Collect bp hr min 2 5kb protocol 5 10 kb protocol 2 000 3 000 4 000 E ES 8000 19000 Run times are affected by temperature and broad range collections Times should be used as an approximate guideline
28. d be used when collections that are broader than the minimum are required For size reference either internal DNA standard or external DNA markers run in a separate lane may be used depending on cassette type and or application Cassette kits for smaller DNA size range collections 90 1500 bp use internal standards and larger collections gt 2 kb require use of one cassette lane to run and external reference marker Assignation of reference DNA is detailed in Sections 6 5 and 6 6 In the protocol editor 1 Select an appropriate cassette type for size selection if a new protocol is being created 2 Assign the internal or external reference DNA a For cassettes using internal standards I press the USE INTERNAL STANDARDS button b For cassettes using an external marker lane I enter the lane into which the marker will be loaded into the Reference Lane field 1 is the default value ll press the APPLY REFERENCE TO ALL LANES button 7 Select the Range programming mode Click the Range button for the lane from which the cut will be extracted If using and external marker make sure no collection modes are selected for that lane 8 Enter a range values in the BP Start and BP End fields The median target value of the collection range will auto fill the BP Target field 9 Optional Enter a BP pause value If a value is entered into the pause field the system will pause w
29. e Tight Targets I l 00 30 00 40 Time hh mm Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 100 600 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 3 888 744 2244 www sagescience com support sagescience com 24 3 2 Agarose Dye Free 100 bp 600 bp Marker X1 internal standards 25 ul elution well Use Product Number BSF2010 Use Cassette Definition 2 DF Marker X1 low volume DNA Marker X1 Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets l l 00 30 00 40 Time hh mm Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 100 600 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 4 888 744 2244 www sagescience com support sagescience com 24 4 2 Agarose Dye Free 100 bp 600 bp Marker M1 external marker Use Product Number BEF2010 Use Cassette Definition 2 DF Marker M1 DNA Ma
30. e selected DNA Sample 40 tl Buffer 30 pl Sample 40 ul Figure 11 1 An illustration of the electrophoretic effect ona DNA sample when a sample well in not completely filled BluePippin Operations Manual software v 6 13 cassette definition Set 15 11 1 888 744 2244 www sagescience com support sagescience com 1 Re check the buffer level in the sample wells Make sure that sample wells are completely full to the top with electrophoresis buffer Top off with additional buffer if necessary The total volume of the sample well is 70 ul Remove 40ul of buffer from the first sample well and load 40ul of sample or marker into that well Take care not pierce the agarose with the pipette tip There is gel on all sides and bottom of the sample well In addition there is an agarose chimney surrounding the top of the sample well that protrudes up through the cassette cover see Figure 11 1 When removing buffer some users find it useful to immerse the pipette tip just below the surface of the buffer and follow the liquid level down with the tip as the buffer is removed When buffer removal is completed there will be 30ul of buffer left in the well When adding sample place tip of pipette just below the surface of the buffer and follow the liquid level up with the tip as the well fills Don t be concerned if the sample well slightly overfills The density of the sample will allow it to sink before it can flow out of
31. e left anda target directory portable flash media i e USB key on the right File Manager Tab Pippin Destination File Drive File Directory irorenmn ron I lo Directory Pippin Destination Files Drive Files UNMOUNT FLASH DRIVE File Types File Commands BluePippin Operations Manual software v 6 13 cassette definition Set 15 19 1 888 744 2244 www sagescience com support sagescience com 18 2 File Types GO TO LOGS GO TU PROTOCOLS GO TO CASSETTES There are three types of files that are stored in different directories Log Files The log file directory is accessed by pressing the GO TO LOGS button Every run on the BluePippin automatically saves the following files which are saved with the following convention software version _ year month day _ hour minute second _ user input protocol name or test type file extention e Log File This is a text txt file that contains all of the data that was collected during arun This file can be used as a diagnostic tool with Sage Science support personnel Data from the log file can be viewed in the Log Review Screen using the PC Pippin Review application or copied into a spreadsheet file to recreate the data displayed in the BluePippin graph images e Screen Image File An image file png of the Main Tab screen at the completion of the run is saved The log and screen image files e Continuity Test After every test an image file png is aut
32. ection 14 3 for details Collection ranges 10 5 CV 6 6 CV Input gDNA 5 ug 5 0 CV Tight Figure 4 1 A comparison of the relative sample yield between tight and broader range size selections of DNA BluePippin Operations Manual software v 6 13 cassette definition Set 15 4 1 888 744 2244 www sagescience com support sagescience com 4 2 2 DNA undergoing elution is smaller than DNA at the detector The branch point between the separation and elution channels is downstream from the detector position Figure 4 2 shows a cassette channel to illustrate During normal operation the leading edge of the DNA fraction scheduled for elution passes the detector before the start of elution by up to several minutes This offset can give rise to the impression that sample elution is late even in runs that are functioning properly LED Detector Branch Point DNA Migration Figure 4 2 An illustration of the time and base pair difference between the detector and branch point 4 2 3 The rate of electrophoresis is faster in the separation channel than in the elution channel This may cause misconceptions with regard to the timing of broad size selections For instance if one sample is programmed to select from 400 600bp and a second sample is programmed to select from 200 600 bp the narrower range will finish eluting before the broader one even though both elutions complete collection at 600 bp The collect
33. ed of according to internal lab safety policy Cassettes should be disposed of as laboratory waste O Note Dye free cassettes fluorescein dye bound to the reference marker BluePippin Operations Manual software v 6 13 cassette definition Set 15 21 1 888 744 2244 www sagescience com support sagescience com 22 Ordering Information Catalogue No BluePippin Includes monitor keypad mouse and accessories BLU0001 Dye Free Gel Cassettes with Internal Standards 10 pk Internal Catalogue Standards No 3 agarose dye free 90 250 bp Q2 BDF3010 2 agarose dye free 100 600 bp VI BDF2010 2 agarose dye free 100 600 bp 25 ul elution well XI BSF2010 1 5 agarose dye free 250 1500 bp R2 BDF1510 Dye Free Gel Cassettes with External Marker 10 pk External Catalogue Marker No 0 75 agarose low range 1 10kb SI BLF7510 0 75 agarose upper range 10 18 kb U1 BUF7510 0 75 agarose mid range 18 27 kb T1 BMF7510 High Range Collections 40 or 50 kb WI BHF7510 Gel Cassettes Band Capture 10 pk External Catalogue No Marker 1 5 agarose low range band capture 250 1500 bp A 0 75 agarose low range band capture 2 10 kb D BLG7510 2 agarose dye free 100 600 bp BEF2010 The elution volume in other cassettes is 40ul BluePippin Operations Manual software v 6 13 cassette definition Set 15 22 1 888 744 2244 www sagescience com support sagescience com Ordering Infor
34. em Options La n e Status Sample ID Current Elution Timer Reference Idle Separate Elute 4 0 00 00 00 00 1 P d z 0 00 00 00 00 1 P i OO o eco ae oor r_ 1n k1n1 mml h tm Protocol 00 00 00 15 00 30 00 45 01 00 01 15 01 30 01 45 02 00 02 15 02 30 Time hh mm St al U S Protocol Name P protocol xyz ane Progress hh mm ss Voltage Y Clock 99 9 07 Feb 2012 17 07 31 Remote Access Estimated Completion Time 07 Feb 2012 18 06 49 Control TM REZZA AAA Panel 0000 0015 0030 0045 0100 0115 0130 0145 0200 0215 02 30 Rs eae CALIBRATE SNAPSHOT INFO SHUTDOWN If switching from a Midori Green cassette to a dye free cassette rinse the electrode array by filling the rinse cassette with water placing the rinse cassette onto the optical nest and closing the lid for 20 seconds Press CALIBRATE on the Control Panel TEST START CALIBRAT SNAPSHOT INFO SHUTO i BluePippin Operations Manual software v 6 13 cassette definition Set 15 9 1 888 44 2244 www sagescience com support sagescience com 3 An LED Calibration window will launch The Calibration Status field will contain the message Calibration not done Place fixture on the optical nest so that all five LED detectors are covered The dark side of the fixture must be down closest to the LEDs Top Bottom Correct Position on Optical Nest BluePippin Operations Manual softwa
35. ent replacement kit for 10 cassettes Marker A1 RGL1510 BluePippin Operations Manual software v 6 13 cassette definition Set 15 22 2 23 888 744 2244 www sagescience com support sagescience com Instrument Specifications Specifications for BluePippin Instrument Electrophoresis Voltage 25V 100V 150V constant 100 V pulsed Optical detection 470 nm excitation 525 nm emission Power Requirements 100 240 VAC 10 VAC 2 5 A 50 60 Hz Weight 15 lbs 7 kg Dimensions THX 11W X 21D in 18H X 28W X 53D cm Approvals CE CSA Country of Origin United States BluePippin Operations Manual software v 6 13 cassette definition Set 15 23 1 888 744 2244 www sagescience com support sagescience com 24 Gel Cassettes Specifications Specifications Dye free cassettes Minimum Sample Load low single nanograms Maximum Sample Load 2 ug sheared genomic DNA Reproducibility specified on following pages Sample Recovery 50 80 1 Upto 10 ug of DNA may be loaded however accuracy may be affected by load amounts over 2 ug and the distribution profile of DNA fragments See www sagescience com support for instructions on using higher amounts of DNA Deviation of actual target value from software input value divided by the actual value 2X standard deviation of replicate samples Recovery is measured using known amounts of plasmid marker ladder EIN Sample Recovery 50 80 5 2X standard deviation of replicate
36. equired to program a protocol Detailed instructions are provided in the subsequent sections 1 Click the Protocol Editor tab along top of the screen 2 Select NEW to create a new protocol To edit an existing protocol press the LOAD button to select the protocol to be edited Protocol Changes NOT Applied amp SAVE AS 3 Select a Cassette Type from Cassette menu by clicking the folder icon Protocol fields cannot be edited unless a cassette type has been selected BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 2 888 744 2244 www sagescience com support sagescience com 4 Make sure the End Run when Elution is Completed box is checked This will end the run at the earliest possible time after the final elution is complete Run Time hh mm End Run when Elution is Completed x O Information The 8 hour run value is a default value that ensures most runs will be completed if the End Run when Elution Completed box is left unchecked 5 Program the size selection protocol and assign the DNA reference marker lane There are four modes of programming on the BluePippin e Tight collects minimum allowable distribution range of DNA fragments using the median target base pair value e Range allows users to select the range to be collected using starting and ending base pair values e Time allows users to program extractions using the starting a
37. es can be used directly for ligation and amplification without buffer exchange Oo oP Bb BluePippin Operations Manual software v 6 13 cassette definition Set 15 13 1 888 744 2244 www sagescience com support sagescience com 14 Sample Recovery and Yield 14 1 Overview For most DNA collections the intrinsic sample recovery based on the input and recovery of restriction fragments is between 50 80 section 14 2 The amount of DNA recovered from a sheared fragmented sample is dependent on the input DNA distribution profile where the size selection occurred relative to that profile and the range of fragments collected section 14 3 DNA may become entangled in the ultra filtration membranes in the elution modules which can reduce yield The BluePippin automatically adds a brief field reversal at the end of each elution to push DNA off of the membrane Section 14 4 However for higher molecular weight DNA collections gt 2kb we recommend several user steps to help improve sample recovery section 14 5 14 2 Intrinsic Sample Recovery on the BluePippin Intrinsic DNA recovery in BluePippin cassettes is determined by running known amounts of a plasmid restriction digest on the BluePippin collecting a broad range of fragments from the digest and comparing the input and product profile quantitatively using the Agilent Bioanlyzer 2100 The intrinsic recovery of DNA fragments for most Pippin cassette types is between 50 80 F
38. escience com support sagescience com 14 5 HMW DNA Recommendations to Improve Yield Sage Science recommends the following procedures in order of effectiveness to maximize the yield for high molecular weight DNA gt 2kb and high pass protocols 1 Wait atleast 45 min after termination of the run before removing eluted DNA it is OK to leave samples in instrument for hours i e for an overnight run 2 Additional 10 30 yield can often be achieved by rinsing the elution well with TE 0 1 Tween 20 Tween solution is provided by Sage Science with cassette kits for HMW collections After removing the initial eluted volume Remove eluted sample from elution well at the end of the run Add 40ul Tween solution supplied with the cassettes to the elution well Wait 1 minute Remove solution and pool with the original extracted sample or process separately 3 When removing elution well contents mix the solution gently by pipetting up and down several 5 to 10 times before removing BluePippin Operations Manual software v 6 13 cassette definition Set 15 14 5 15 888 744 2244 www sagescience com support sagescience com System Validation Control DNA can be purchased from Sage Science to check the performance of the BluePippin system The Control DNA is useful to test refine and troubleshoot BluePippin size fractionation protocols For comparison to factory tested cassettes an extraction protocol a typical Agilen
39. hen it has estimated that that base pair value has been collected during the elution of that lane A pause will allow a user to remove two consecutive cuts from a sample The two cuts will usually have overlapping size distributions User may wish to use the pause feature for two reasons 1 to retrieve nearly identical cuts from the same sample or 2 to avoid overflow of the elution module during collections that require a long elution period The BP pause value must be within the extraction range between BP Start and BP End The pause function temporarily turns off power to the electrodes and suspends run timers allowing users to remove the sample rinse the elution well if desired and then resume the instrument run to collect additional sample Important A pause will require the user to manually resume the protocol from the Main Screen controller The instrument lid may be opened during the pause and sample retrieved BluePippin Operations Manual software v 6 13 cassette definition Set 15 1 3 888 744 2244 www sagescience com support sagescience com Enter Sample ID information 10 Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new press Save If a protocol has been edited a yellow alert will be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As will al
40. igure 14 1 shows the recovery of a 5kb band from a 0 75 cassette 59 Recovery Blue input DNA1 2 dilution Red extracted DNA 1 1 dilution IR f 11 l ER Bioanalyzer Standard 1500 2000 10380 amp Lancs 6 1 2Ka0 500 dithnofrou Figure 14 1 An example of the intrinsic recovery of 5 kb band on a 0 75 agarose cassette BluePippin Operations Manual software v 6 13 cassette definition Set 15 14 2 888 744 2244 www sagescience com support sagescience com 14 3 Improving Product Yield by Selecting a Wider Size Range The BluePippin can produce very narrow size distributions from sheared genomic DNA However narrower size distributions will necessarily mean that a smaller fraction of the input DNA will be recovered Users should broaden their collection ranges if the default tight settings do not produce enough DNA for their application Figure 14 2 shows an example of the effect of size range selection vs yield Collection ranges 10 5 CV f 6 6 CV Input gDNA 5 ug Ef 5 0 CV Tight ee Aan jl ue Spe a Bioanalyzer Result Average nd Size bp BP Start BP End Figure 14 2 Increasing yield by widening the size selection range D Information The per cent recovery specification is determined by collecting known amounts of restriction fragments Section 10 2 Product yield from a sheared DNA sample is influenced by the fragment distribution and input
41. in flat bubble between the gel column and the bottom of the channel If this happens in the region used by the optical detector see illustration DNA detection will be extremely unreliable If a lane with such a bubble is used for the reference marker the markers will not be properly identified and sample collection will occur at the wrong time or fail altogether Only the optical properties of the channel are affected electrophoretic properties of the channel are unaffected since the DNA travels through the center of the gel column above the bubble To find such bubbles turn the cassette upside down and view the bottom of the channels under a strong light while tilting the cassette back and forth see Figure 10 2 below Figure 10 3 shows the region of optical detection Important f a bubble is observed do not use the lane to run the reference marker or for peak capture mode applications It may be used for size selections using an external marker in a different reference lane tight or bp range modes AM Important Flat bubbles between the top surface of the gel column and the plastic top of the channel will NOT affect run quality or optical detection They may be used for markers or sample without any adverse effects Agarose Delamination Optical Region Do not use this lane for samples with internal standards or external DNA marker Figure 10 2 Bubble in optical path Do not use the lane for the DNA reference marker
42. ions of large DNA fragments especially with pulsed field protocols can also appear counter intuitive with respect to timing Rate of Migration Separation 25 Faster Elution Figure 4 3 An illustration comparing the relative rate of electrophoresis in the separation and elution channels BluePippin Operations Manual software v 6 13 cassette definition Set 15 4 2 888 44 2244 www sagescience com support sagescience com BluePippin Operations Manual software v 6 13 cassette definition Set 15 4 3 9 888 44 2244 www sagescience com support sagescience com Sample Preparation Input Sample Characteristics When running the BluePippin characteristics of input DNA can affect separation resolution and efficiency of product recovery The following general guidelines should be followed lonic strength The ionic strength of the sample should be lower than the ionic strength of the buffer 80mM monovalent ions High salt concentrations can result in slower than expected DNA mobility Protein in the sample DNA binding proteins such as ligases or polymerases can affect the mobility of fragments during separation Proteins can also reduce DNA recovery from the elution module by increasing the binding of DNA to the ultrafiltration membrane at the back of the elution module For best results samples should be de proteinized prior to loading whenever possible Input DNA size distribution A knowledge of the input s
43. ippin extracts narrow or wide DNA size distributions more reproducibly and with higher yield than manual preparative gel techniques However due to the unique design of the pre cast gel cassette there are key differences between running the Pippin and other agarose gel methods 4 1 Keys to Successful Collections It is recommended that users closely follow the instructions outlined in the following sections Section 5 14 until they become thoroughly familiar with the system The Quick Guide included with cassettes provides protocol reminders but is not intended for beginning users The keys to successful collections are Purity of the input sample Section 5 Optical Calibration Section 9 Preparation of the cassettes Section 10 Sample loading Section 11 In addition for High Molecular Weight DNA collections e Recommendations to improve sample yield Section 14 5 4 2 Common Misconceptions Users should be aware of the following characteristics of the BluePippin System These are part of normal operation of the system but may seem counterintuitive at first 4 2 1 Narrowest is not always the best The BluePippin can produce very narrow size distributions from sheared genomic DNA However narrower size distributions will necessarily mean that a smaller fraction of the input DNA will be recovered Users should broaden their collection ranges if the default tight settings do not produce enough DNA for their application See S
44. ize distribution is obviously important to program accurate size selection settings BluePippin cassettes are calibrated using the Agilent Bioanalyzer to evaluate input and product sizes and so for best results input size distributions should be evaluated using the Bioanalyzer For low concentration samples the Agilent HS chip is very useful Preparing DNA Samples for the BluePippin PER Bring DNA sample up to 30ul with TE Bring loading solution to room temperature For each sample combine 30ul of DNA sample with 10ul of loading solution Mix samples thoroughly vortex mixer Briefly centrifuge to collect Recommended sample Load Guidelines Maximum Load 10ug sheared genomic DNA 2 ug restriction PCR band capture Minimum Load low single nanograms sheared genomic DNA 50 ng restriction PCR band capture Optical Sensitivity approx 10 15ng single bands restriction Midori Green cassettes fragments PCR products or synthetic DNA fragments BluePippin Operations Manual software v 6 13 cassette definition Set 15 5 1 888 744 2244 www sagescience com support sagescience com 6 Protocol Programming Guide The default screen on the BluePippin is a tabbed format The Protocol Editor is the second tab 6 1 Overview The protocol editor tab is the screen with which users create new protocols or edit existing protocols for DNA size selection A protocol is created for a cassette type within the applicable target ra
45. low a new name for the file to be applied Protocol Changes NOT Applied 4 SAVE AS All protocol files are saved in a directory nome pippin BluePippin Protocols and may be accessed in the File Manager tab 7 3 Time Mode programming timed collections M Important The time mode does not reference a standard Cassette to cassette variablity and environmental running conditions will cause a wide variation in selection accuracy The Range programming mode is a method for crude DNA collections where accuracy of the fragment size or range is not critical The system will collect the fragment range between the elution start time and elution end time In the protocol editor 1 Select the Time programming mode Click the Time button for the lane from which the cut will be extracted A DNA marker reference standard is not required 2 Entera time values in the T Start and T End fields Sample elutions will occur at exactly those times after the start of the run 3 Optional Enter a Time pause value lf a value is entered into the pause field the system will pause at that time after the start of a run A pause will allow a user to remove two consecutive cuts from a sample The two cuts will usually have overlapping size distributions User may wish to use the pause feature for two reasons 1 to retrieve nearly identical cuts from the same sample or 2 to avoid overflow of the elution module during collections
46. ly seated into the bottom of the nest Detection of DNA within the cassette will fail if the bottom of the cassette is not properly seated against the optical region 3 Remove adhesive strips from cassette Place one hand on the cassette and hold it firmly in the nest Grab the white tabs of the tape and pull the strips firmly and slowly toward the front of the BluePippin until they are removed 4 Remove buffer from elution modules and replace with 40ul of fresh electrophoresis buffer Replenishing the buffer ensures proper electrophoretic continuity When refilling empty modules place tip all the way into the module and slowly fill from the bottom withdrawing the pipette tip while dispensing and taking care not to generate any bubbles that can occlude the current path Use spare buffer that has been supplied with the cassette package or pipette from the buffer chambers BluePippin Operations Manual software v 6 13 cassette definition Set 15 10 3 888 744 2244 www sagescience com support sagescience com Important Make sure that the pipette tips used for step 4 extend all the way to the bottom of the elution modules without sealing the elution port opening If the tips seal the port opening it will be extremely difficult or impossible to empty and refill the elution module completely Test tip fit using the empty rinse cassette supplied with the instrument Note The total volume of the elution module when filled to the top is 65ul
47. mation Cont d Control DNA for Performance Validation Catalogue No Control DNA for 3 Gel Cassette Int Std 90 250 bp 5 ug load 16 loads CIS3004 Control DNA 2 Gel Cassette Int Std 100 600 bp 5 ug load 16 loads CIS2004 Control DNA for 2 Gel Cassette Ext Mkr 100 600 bp 5 ug load 16 loads CON2004 Control DNA for 1 5 Gel Cassette Int Std 250 1500 bp 2 ug load 16 loads CIS1504 Control DNA for 0 75 Gel Cassette Ext Mkr 1 10 kb 5 ug load 16 loads CON7504 Control DNA for 0 75 Gel Cassette Ext Mkr 10 27 kb 5 ug load 16 loads CON7604 Control DNA for 0 75 Band Capture Ext Mkr 1 kb fragment 5 ug load 4 loads CBC1501 Control DNA for 0 75 Band Capture Ext Mkr 3 kb fragment 5 ug load 4 loads CBC7501 Control DNA for 0 75 Gel Cassette Ext Mkr 10 kb fragment 5 ug load 4 loads CBC7601 Reagent Replacement Kits Dye Free with Internal Standards Catalogue No BDF3010 reagent replacement kit for 10 cassettes Marker Q2 RBF3010 BDF2010 reagent replacement kit for 10 cassettes Marker V1 RBF2010 BSF2010 reagent replacement kit for 10 cassettes Marker X1 BDF1510 reagent replacement kit for 10 cassettes Marker R2 RBF1510 Reagent Replacement Kits Dye Free with External Marker Catalogue No Reagent Replacement Kits Band Capture External Marker Catalogue No BLG7510 reagent replacement kit for 10 cassettes Marker D1 RGL7510 BLG1510 reag
48. n Operations Manual software v 6 13 cassette definition Set 15 2 1 888 744 2244 www sagescience com support sagescience com Unpacking and Installation Unpacking the BluePippin The BluePippin Instrument The BluePippin instrumentation is shipped in two boxes one will contain the BluePippin and Accessories and the second box will contain the computer monitor in the manufacturer s original packaging With the boxes in the upright position open and confirm that the following items are enclosed Monitor e LCD computer monitor e Video cable e Power cord BluePippin e BluePippin Instrument e Accessory box o Computer keyboard USB Computer mouse USB Power supply Plug adapter adapts non U S power cords see Figure 3 1 Power cord Rinse cassette for maintenance of electrodes Calibration cassette for setting optical baseline before each run O O O Or OO o f IEC320 C14 IEC320 C5 Figure 3 1 Adapter plug for non US power cord installation BluePippin Operations Manual software v 6 13 cassette definition Set 15 3 1 888 744 2244 www sagescience com support sagescience com Setting up the BluePippin 1 Remove the accessory box located on inside edge of box at the front end of the instrument Remove the keyboard mouse and instrument power supply from their packaging Firmly grip both sides of the instrument and lift it from the foam packaging insert The BluePippin weighs approximately 14 lb
49. nd ending elution time hr min sec values only a reference DNA marker is not used e Peak collects the next peak restriction fragment or PCR band after the set threshold base pair value has been reached Tight O Note Programming modes may be independently applied to individual sample lanes 6 Enter Sample ID or description optional BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 3 888 744 2244 www sagescience com support sagescience com 7 Press Save As button enter a name for the protocol 6 3 Selecting a Cassette Definition Cassettes are selected from a menu tree with the up to 3 tiers Users should select the appropriate definition from the 2 or 3 tier e Agarose the percentage agarose of the cassette 3 1 5 0 75 o Range lf the same type of cassette requires a different electrophoresis protocol the cassette will be selected by target size range and the type of electrophoresis protocol 1 6 kb Low Voltage 1 6 kb Pulsed Field 3 10 kb Pulsed Field 13 33 kb Pulsed Field 20 50 kb Pulsed Field O O O 0 O 0 75 DF 2 6kh Marker 51 0 70 DF 310kh Marker 51 Ofo DF Low Voltage 1 Gkb Marker 31 EAP AND ALL COLLAPSE ALL CANCEL BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 4 888 744 2244 www sagescience com support sagescience com 6 4 Run Times Protocols can be run with widely variable r
50. ned Some BPstart or BPthresh values out of ladder range BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 9 888 744 2244 www sagescience com support sagescience com 7 Programming Size Selections Tight Range and Timed Modes 7 1 Tight Mode programming minimum size range collections The Tight programming mode is a method for selecting DNA size range collections by entering a target base pair value in software The system will collect the minimum fragment distribution that is possible centered on this base pair value For size reference either internal DNA standard or external DNA markers run in a separate lane may be used depending on cassette type and or application Cassette kits for smaller DNA size range collections 90 1500 bp use internal standards and larger collections gt 2 kb require use of one cassette lane to run and external reference marker Assignation of reference DNA is detailed in Sections 6 5 and 6 6 In the protocol editor 1 Select an appropriate cassette type for size selection if a new protocol is being created 2 Assign the internal or external reference DNA a For cassettes using internal standards i press the USE INTERNAL STANDARDS button b For cassettes using an external marker lane i enter the lane into which the marker will be loaded into the Reference Lane field 1 is the default value ii press the APPLY REFERENCE TO A
51. nge and is saved as a named protocol file with a ppprot file extension When running a cassette an appropriate protocol file is selected and applied to the run A protocol file may be applied to any run provided the cassette type is correct Protocol Editor Tab Cassette us Type ame Selector Run Time End Selector Protocol Parameters DNA Marker Standards Warnings Lane Assignment a l Text Box File ef Commands BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 1 888 744 2244 www sagescience com support sagescience com Protocol Parameters Detail The protocol parameters section is shown below Users will select a programming mode for each sample and a lane to designate as a reference Then the size selection values are entered either as base pair BP values or time values hr min sec in Time mode For Peak programming mode users enter a base pair value into the BP Thresh field the instrument will collect the next peak that it detects after that threshold has been reached Individual lanes may have different parameters and different modes Base Pair BP Programmin Ref Lane Tight and Modes i Indicators pe wee ein alles parameters P parameters Tight Range Time Peak Ref Lane BP Target BP Start BP End BP Pause T Start T End TPause BP Thresh Stat Exp End Exp Sample ID Template 6 2 Programming Summary The following is a Summary of the steps r
52. nt Software 1 Insert the USB drive with the upgrade folder into the USB port on the front panel of Pippin instrument 2 From the Main Tab press INFO This will launch the information window TEST START CALIBRATE 1 SNAPSHOT INFO SHUTOO TH 3 From the Information Window press SOFTWARE UPGRADE Sage Science 500 Cummings Center Suite 3150 Beverly MA 01915 USA support sagescience com Blue Pippin Instrument Name Worm Instrument ID PXOO SW Version 5 30 Build Date 24 Feb 2012 Fi Version 3 62 Address 127 0 0 1 WAC Address 00 30 18 AF 34 A3 UDP Server Port 50000 USER MANUAL SOFTWARE UPGRADE 2012 Sage Science http fsagescience com Parts 2012 Solidus LLC http solidusile com Parts 2012 National Instruments httpini com 4 Press OK at the warning prompt the Software Upgrade Window will launch Warning SELECT OK TO EXIT THE MAIN PROGRAM AND LAUNCH UPGRADE UTILITY BluePippin Operations Manual software v 6 13 cassette definition Set 15 20 3 888 44 2244 www sagescience com support sagescience com 5 Press OK at the warning prompt the Software Upgrade Window will launch New Version Flash Drive 16 Jul 2010 15 35 000 14 Oct 2010 16 15 Lott 16 Jul 2010 13 00 Lee 14 Oct 2010 13 00 Lay DBA1E0D753B4A069 EE1CB198FA72DB25 stem ready for upgrade Check version numbers ti make sure that an upgrade is required if yes select INSTALL UPGRADE Otherwi
53. nthe protocol editor enter the reference lane designation into Ref Lane field for every cassette lane Click APPLY REFERENCE TO ALL LANES Figure 6 1 shows the proper settings for running a DNA marker in lane 1 with programmed Tight extractions in lanes 2 5 Lane to which marker has reference off of marker lane Reference Lane L APPLY REFERENCE TO ALL LAMES Remaining lanes will USE INTERNAL STANDARDS Tight Range Time Peak RefLane Figure 6 1 The correct configuration for a protocol with an external marker loaded in lane 1 BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 6 888 744 2244 www sagescience com support sagescience com 6 6 Assigning Reference Markers internal standards 1 Markers must be loaded into every lane with the sample 30 ul of sample with 10 ul of internal standard loading solution 2 Inthe protocol editor press USE INTERNAL STANDARDS This will auto fill the Ref Lane fields with reference marker internal to the sample The software will determine the timing of elution based on the migration of the DNA marker within its own lane 3 Figure 6 2 shows the correct assignation of marker lanes for internal standard runs Reference Lane All lanes will reference off of USE INTERNAL STANDARDS internally loaded standard APPLY REFERENCE TO ALL LANES Tight Range Time Peak RefLane Figure 6 2 The correct configuration for a pr
54. omatically saved e Calibration Test After every test an image file png is automatically saved In addition a screenshot may be manually saved as at any time during a run as a png file by pressing Snapshot in the Main Tab on the controller The same file convention is used but with the word Screenshot in the file name PAUSE STOP SNAPSHOT BluePippin Operations Manual software v 6 13 cassette definition Set 15 19 2 888 744 2244 www sagescience com support sagescience com Protocol Files The protocol file directory is accessed by pressing the GO TO PROTOCOLS button Protocol files have a ppprot extension and are stored in a Protocols directory with the name saved by the user Protocols may be copied to a flash drive for archiving or transfer to another BluePippin Cassette Files The log file directory is accessed by pressing the GO TO CASSETTE button Cassette files have a ppcass extension and contain the calibration parameters for the cassettes that are indicated in the file names Cassette files may be copied to a flash drive and transferred to another BluePippin Cassette files are not editable Note Cassette calibration files may be updated during software upgrades Users should check release notes and save legacy cassette files if a methods have been developed with their use Managing Files The commands for managing files are found at the bottom of the File Manager Tab The functi
55. ons are outlined below NEW FLASH FOLDER UNMOUNT FLASH DRIVE Creates new Copies Copies folder on highlighted highlighted Nu ua attached USB file s from file s from files flash drive Flash drive Pippin to flash drive prior to flash drive to Pippin removal A Warning Be sure to Unmount Flash Drive prior to removal Failure to do so may cause data loss Failure to do so may cause data loss and problems with future upgrade procedures BluePippin Operations Manual software v 6 13 cassette definition Set 15 19 3 888 744 2244 www sagescience com support sagescience com 20 Upgrading Software Pippin upgrade software is available for download at www sagescience com support The files are provided in a zip file and the contents must be extracted to the root directory of a USB flash drive 20 1 Extracting the Files to a USB flash drive 1 Download the Software Upgrade package by pressing the appropriate link on www sagescience com support in the Downloads section under Software 2 Press Save File when prompted The zip file will likely save to a Downloads folder on your computer The file will be named pippinupgrade version number zip 3 Copy the zip file to a USB drive Make sure the file is transferred to the root directory and not placed within a folder 3 4 Right click on the zip file and select Extract All Mame Date mod E Pippinupgrdele sas Open Open in ne
56. otocol using internal standards N Important When using internal standards all samples must be prepared the BluePippin internal standard loading solution which is formulated for the cassette type and size range to be used The internal standard loading solution contains the markers BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 7 888 744 2244 www sagescience com support sagescience com 6 7 Warnings and Indicators BP Range Flag When a Minimum Size Range has been programmed into a sample lane the BP Range Flag field for that sample will display tight and a green color BP Range Flag When a broad size cut range larger than the minimum has been programmed into a sample lane the BP Range Flag field for that sample will display broad and an orange color BP Range Flag Pause Enabled Indicator If the pause feature has been entered for a sample lane Pause On indicator will be displayed Pause On BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 8 888 744 2244 www sagescience com support sagescience com Warnings Text Box If there are errors or exceptions within a programmed protocol a message will appear in the Warnings text box identifying the nature of the discrepancy Warnings Some elution ranges will cause chamber overflow Some start values missing Some end values missing Some start fend values ill defi
57. ove the sealing tape strip that covers the elution wells Samples can be removed from elution modules using a standard 100 200uI pipette Important Make sure that the pipette tips used for collection extend all the way to the bottom of the elution modules without sealing the elution port opening If the tips seal the port opening it will be extremely difficult if not impossible to completely recover the contents of the elution module Test tip fit using the empty rinse cassette supplied with the instrument gt Dr 3 Samples from standard cassettes without sealed elution modules will be recovered in approximately 40ul of electrophoresis buffer 4 When all samples are collected remove used cassette from instrument and dispose of it properly Important Eluted samples should not be left in the cassette for longer than a couple of hours to avoid poor recovery due to adsorpsion or diffusion Important Used cassettes should never be allowed to sit in closed instruments for long periods of time e g overnight Under those conditions humidity from the cassette reservoirs can accelerate corrosion of the electrode assembly located in the sliding cover Information Electrophoresis buffer is 50 mM Tris 30 mM TAPS 0 1 mM EDTA During elution EDTA does not rapidly equilibrate across the ultrafiltration membrane of the elution module and so the final concentration of EDTA in eluted samples is elevated to 1 2 mM Information E uted sampl
58. ovides the narrowest distribution that is Time hh mm obtainable and Broad provides a wider distribution around the 50 kb target for a higher product recovery Estimated Collection Times Add Target hr min min Target 50kb tight 50kb broad Minimum Size 5 Distribution as CV 10 14 Reproducibility Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 15
59. placement On site maintenance or repair is not implied Do not attempt to repair or disassemble the BluePippin instrument This will void the remaining warranty and possible create a dangerous condition Sage Science support staff may ask for a log file or image file to evaluate or troubleshoot instrument issues High Voltage The BluePippin contains high voltage apparatus The instrument should be repaired by authorized personnel only Do not disassemble under any circumstance Cassettes Cassettes have a 1 year warranty If a cassette or sample lane is defective Sage Science will replace the cassette at no cost The defective cassette should be disposed of as per regulations at the user s facility Sage Science support staff may ask for a log file or image file to evaluate or troubleshoot cassette issues Maintenance It is possible that salts will accumulate on the electrodes that are housed in the BluePippin lid assembly This may alter the performance of the instrument over time It is recommended that the electrodes undergo periodic rinsing To rinse the electrodes use the rinse cassette provided in the instrument shipment fill with deionized water load on the sample tray and close the BluePippin lid A quick rinse for several second is sufficient If a rinse cassette is lost or damaged contact Sage Science support for a replacement No other maintenance activity is required Cassette Disposal Reagents and buffer should be dispos
60. profile of the sample BluePippin Operations Manual software v 6 13 cassette definition Set 15 14 3 888 744 2244 www sagescience com support sagescience com 14 4 Improving Product Recovery with the Field Reversal The BluePippin provides a brief current reversal at the end of a sample elution can improve sample recovery by up to 20 The field reversal is a default setting located in the System Options Tab of the Pippin Software The use of this option is recommended but may turned off by users Figure 14 3 below shows the improvement that can be achieved Figure 14 4 shows the setting option in software When activated the setting will be applied all subsequent runs until the user again changes the settings in the System Options Tab 2 cassette pBR 322 Mspl digest 40 min elution With current reversal ALT 0 Cogo Without current reversal 200 300 400 500 Size of Fragment Bp Figure 14 3 Improving recovery with a 5 sec current reversal A 40 minute elution of a plasmid restriction digest was run and each data point represents an individual band Instrument Mame POO Use Reverse Field for Sample Recovery Use this button to turn field reversal OFF ON and then press Accept The default setting is ON Figure 14 4 System Options Tab with expanded view of Reverse Field Option BluePippin Operations Manual software v 6 13 cassette definition Set 15 14 4 888 744 2244 www sag
61. r Midori Green dye cassettes BluePippin Operations Manual software v 6 13 cassette definition Set 15 12 1 888 744 2244 www sagescience com support sagescience com Enter 0 60 for dye free runs Enter 0 30 for Midori Green runs LED Calibration Targetlph mA 5 Press CALIBRATE 6 The Calibration Status field will contain the message Calibration OK Press EXIT 7 If necessary select the protocol to be run from the Protocol Name drop down menu in the Protocol Status Panel The last protocol that has been programmed will automatically be loaded See Chapter 11 Writing and Editing a Protocol for instructions on programming Protocol Name protocol xyz Progress hh mm ss Voltage Clock 99 9 07 Feb 2012 17 07 31 Remote Access Estimated Completion Time 07 Feb 2012 18 06 49 8 Loada properly prepared cassette Chapter 10 into the optical nest Typically users will load samples while the cassette is on the nest see Chapter 11 Ensure that adhesive tape has been placed over the elution wells 9 Press START on the Control Panel TEST START CALIBRATE SMAP SHOT N neo SHUTDOWN BluePippin Operations Manual software v 6 13 cassette definition Set 15 12 2 888 744 2244 www sagescience com support sagescience com 10 An LED Calibration window will launch If the instrument has not been calibrated or if it has been calib
62. rated for the wrong cassette type users have to opportunity to remove the cassette place the optical calibration fixture on the nest and calibrate 11 Press START to continue without calibration LED Calibration Target ph mA Iteration LED Current counts Photocurrent mA 009 Calibration Status Prompt Load TARGET cassette close lid and CALIBRATE To start without calibration load TARGET cassette and START To cancel calibration and protocol CANCEL CALIBRATE TART CANCEL 12 The LED Calibration window will close 13 On the Lane Status Panel the Separate indicators will turn green Section 12 3 14 On the Protocol Status Panel the progress clock will begin and the progress block will appear Section 12 3 BluePippin Operations Manual software v 6 13 cassette definition Set 15 12 3 888 44 2244 www sagescience com support sagescience com 12 3 Monitoring a Run The Lane Status Pane has three indicator lights per lane When the instrument is idle all Idle indicator lights are light gray Reference Idle Separate Elute Sample IC Current Elution Timer Reference Idle Separate Elute D Sample E focoso 1 sampes A 220 Monk 240 CL EEE e Green Separation If a lane is separating fragments the Separate indicator will be green e Orange Elution If a lane is eluting a size range the Elute indicator will be orange and the elution timer will be active
63. re v 6 13 cassette definition Set 15 9 2 888 744 2244 www sagescience com support sagescience com 4 Close the lid 5 To calibrate dye free cassettes the LED Calibration setting Target ph mA must be 0 60 To calibrate cassettes using DNA binding dye Midori Green the LED Calibration setting must be 0 30 Enter 0 60 for dye free runs Enter 0 30 for Midori Green runs LED Calibration Targetl ph mA La ne 5 Pos Lane 4 Lane 3 Lane 2 Lane 1 rx Iteration LED Current counts Photocurrent m oso D 060 Calibration Status Calibration not done Prompt Load desired calibration cassette close lid and CALIBRATE To exit calibration window EXIT CALIBRATE N EAlT 6 Press CALIBRATE BluePippin Operations Manual software v 6 13 cassette definition Set 15 9 3 888 744 2244 www sagescience com support sagescience com 7 TheLED photocurrents will automatically adjust to a factory setting and the Calibration Status field will contain the message Calibration OK LED Calibration Target ph mA Iteration LED Current counts Photocurrent mA coco go ceo D 060 D 060 Calibration Status Calibration OK Prompt To restart calibration CALIBRATE To exit calibration window EXIT CALIBRATE EXIT 8 Press EXIT to return to the Main Tab BluePippin Operations Manual software v 6 13 cassette definition Set 15 9 4 888 744 2244 www s
64. ress Save If a protocol has been edited a yellow alert will be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As will allow a new name for the file to be applied Protocol Changes NOT Applied SAVE AS All protocol files are saved in a directory home pippin BluePippin Protocols and may be accessed in the File Manager tab Important During band collection optical detection is turned off This may cause the detection M of band to appear clipped see image below This appearance is normal and should be expected Typical optical trace from a band capture run BluePippin Operations Manual software v 6 13 cassette definition Set 15 8 3 Graph Display Image Display 888 744 2244 www sagescience com support sagescience com Optical Calibration The BluePippin optical system should be calibrated for the specific type of cassette to be used dye free or Midori Green The instrument is supplied with a calibration fixture that fits into the optical nest If a lab is using only one type of cassette the optics should be calibrated daily when the instrument is in use If users switch cassette type that is to be run e g run a dye free cassette and then a Midori Green cassette or vice versa the optics should always be re calibrated prior to running the new type Main Tab Protocol Editor f Log Review f File Manager i Syst
65. rker M1 Typical times to detector l l l 00 40 01 00 01 20 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets Time to Time to Collect bp min eo EE Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 100 600 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 5 888 744 2244 www sagescience com support sagescience com 24 5 1 5 Agarose Dye Free 250 bp 1 5 kb Marker R2 internal standards Use Product Number BDF1510 Use Cassette Definition 1 5 DF Marker R2 DNA Marker R1 Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets 29 Time to Time to Collect bp min oi w 1200 00 20 00 25 56 Time hh mm 1500 I Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 250 600 bp 800 1000 bp 1200 1500 bp Sample Recovery 50 80 50 80 50 80 Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 6 888 744 2244 www sages
66. s Place the Pippin on a table or bench top Remove the computer monitor from the manufacturer s box and connect the monitor to BluePippin using supplied video cable Connect monitor to power using power supply supplied with monitor Insert USB connector from computer key board into port located in the back of the Instrument Connect mouse to BluePippin via USB or PS2 connector at back of instrument Connect monitor cable into the video port located in the back or the instrument Connect monitor to power using power supply and cords supplied with monitor Connect BluePippin instrument to power via supplied power supply and cable Power connector is at rear of instrument Press power switch located on the rear of the instrument and wait for software to launch approximately 30 seconds The BluePippin is ready for use The software should automatically launch allow 30 seconds Figures 3 2 and 3 3 on the next page show the rear panel of the BluePippin Figure 3 4 on the following page shows the front panel BluePippin Operations Manual software v 6 13 cassette definition Set 15 3 2 888 44 2244 8 www sagescience com support sagescience com USB Ports 4 VGA monitor port Power Switch Figure 3 2 Rear Panel Ports Power Entry Port Key board Monitor Cable Power Cable Figure 3 3 Rear Panel Connections BluePippin Operations Manual software v 6 13 cassette definition Set 15 3 3 888 44 2244 www s
67. samples 6 Recovery is measured using known amounts of plasmid marker ladder Important DNA input samples should be purified to eliminate DNA binding proteins such as ligases polymerases restriction enzymes Bound proteins can alter mobility and reduce recovery Specifications subject to change without notice BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 1 888 744 2244 www sagescience com support sagescience com 24 1 3 Agarose Dye Free 90 bp 250 bp Marker Q2 internal standards Use Product Number BDF3010 Use Cassette Definition 3 DF Marker Q2 DNA Marker Q2 Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets l O0 40 00 50 Time hh mm Run times are affected by temperature and broad range collections Times should be used as an approximate guideline Performance Specifications 90 250 bp Minimum Size Distribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 24 1 for specification definitions BluePippin Operations Manual software v 6 13 cassette definition Set 15 24 2 888 744 2244 www sagescience com support sagescience com 24 2 2 Agarose Dye Free 100 bp 600 bp Marker V1 internal standards Use Product Number BDF2010 Use Cassette Definition 2 DF Marker V1 DNA Marker V1 Typical times to detector Estimated Collection Times for Minimum Size Rang
68. se select UNMOUNT e the flash drive safe FLASH DRIVE to remove the flash dri ely INSTALL UPGRADE UNMOUNT FLASH DRIVE 6 Press INSTALL UPGRADE this will take about 5 seconds INSTALL UPGRADE UN MOUNT FLASH DRIVE 7 Press UNMOUNT FLASH DRIVE UNMOUNT FLASH DRIVE Warning Be sure to Unmount Flash Drive prior to removal Failure to do so may cause data loss and problems with the upgrade procedure 8 Press EXIT to return to Main Menu This will install the software Installation may take up to 30 seconds EXIT 9 To check that the software insturment has been update Press INFO again In the Information window the software version number is listed on the left side Instrument Mame Pipl Sv Version 5 30 IP Addr O Note The system should be ready to use re boot is not necessary BluePippin Operations Manual software v 6 13 cassette definition Set 15 20 4 21 888 744 2244 www sagescience com support sagescience com Warranty and Service Warranty Sage Science will offer a no charge repair or replacement of defective product if notified within the warranty period and product has not been misused altered or damaged by disaster This warranty is not transferable from the original purchaser to a subsequent purchaser Instruments The BluePippin instrument and monitor have a standard 2 year parts and labor warranty Instruments are subject to depot repair or re
69. t Bioanalyzer result is provided with the kits Figure 14 1 shows the DNA profile and extraction protocol for the 2 agarose control kit and Figure 14 2 shows a typical result from a 150 bp tight extraction from the DNA control Kit BluePippin cassettes and instruments are functionally tested using restriction digests of genomic DNA from E coli For each cassette type a different restriction digest is used chosen so that size distribution of the digested DNA closely matches the useful fractionation range of the cassette without any significant peaks or discontinuities Following restriction digestion the control DNA is purified by phenol chloroform extraction dialyzed and diluted into BluePippin electrophoresis The DNA is premixed with BluePippin loading solution and is provided ready for loading no additional loading solution should be added The DNA concentration is 5 micrograms per 40 microliters 40 microliters of control DNA should be used per lane Each tube contains sufficient volume for 16 sample loads Marker bp 20 75 150 300 600 0000 0015 00 30 0045 0100 01145 0130 0145 0200 02 15 on Time hh mm BP End P als3 Figure 14 1 Control DNA profile with reference marker and DNA extraction BluePippin Operations Manual software v 6 13 cassette definition Set 15 15 1 888 744 2244 www sagescience com support sagescience com protocol for a 2 agarose DNA control kit run 50 m Target 150 bp Ave Size 149
70. te cm actenc ss 12 4 Me LOT FIOS siii E S E cain eroici 12 6 SAMPLE COLLECTIONS rr 13 1 dol OV CTO dcr dadi darai 13 1 SAMPLE RECOVERY AND YIELD 2 22 14 2 14 1 OVC VIOW acacia 14 2 14 2 Intrinsic Sample Recovery on the BluePippin serre 14 2 14 3 Improving Product Yield by Selecting a Wider Size Range 14 3 14 4 Improving Product Recovery with the Field Reversal ccccccseeesseeeeeeeeeeeeeeees 14 4 14 5 HMW DNA Recommendations to Improve Yield 14 5 SYTEM VALIDA TON cean E 15 1 SEM OPTIONS TAB ne 16 1 RUNNING IN MANUAL MODE rr rrrrrrrrrei iii 17 1 ANALYZING RUNS LOG REVIEW TAB rr 18 1 MANAGING FILES FILE MANAGER TAB nti 19 1 1b fe 2 02 6 lt 1 eea ee ee ee ee E nee 19 1 19 2 FIE Uy CS alia arie 19 2 UPGRABINGSOPFFWARE cacare en 20 1 20 1 Extracting the Files to a USB flash drive issue 20 1 20 2 Upgrading the Pippin Instrument Software ss 20 3 WARRANTY AND SERV CE 5 2 2 0 2 5 21 1 ORDERING INFORMATION ccccceesseeeeeseeeeeeseeeeenseeeeeneeseeaseeseeaseseeaseeseesseeeneaeenenenens 22 1 INSTRUMENT SPECIFICATIONS ccccceeeseecteseeeeeeneeseenseeeeeneeseenseeseesseseeeneesoensesenenens 23 1 GEL CASSETTES SPECIFICATIONS cccccsesseectesseeseeseeesenseeseenseeeeaseeeoenseesoeneeenennens 24 1 24 1 3 Agarose Dye Free 90 bp 250 bp
71. the Log File field Log File Cid 3 Select a log txt file Select Pippin log file Cancel 2010 10 26 09 39 06 PPLogtxst Ss Help BluePippin Operations Manual software v 6 13 cassette definition Set 15 18 2 888 744 2244 www sagescience com support sagescience com 4 This will populate the Review Log Screen with data from the selected run log Jhome pippin PippinPrep Logs 201 1 05 2011 05 24_10 25 26_tests_2 1 88tight tt Protocol Name Cassette Name un Tim tests 2 188tight 2 Marker B Off BP Target BP Stat BPEnd BP Pause Star ne T Pause BP Thresh Sample ID Template Sig Mon Range Flag Pause Enabled P rotocol 200 T Information DNA Marker Times and Values TEELEC Version Pea und ANALYZE PEAKS SNAPSHOT Optical Electrophoretic i i Current Traces 1908 current drops Traces during elution Visualizing Pippin Run Data The current and optical signal traces are color coded and a key is displayed to the right of the Current graph A check mark next to each lane will toggle the visualization of that lane on and off The type and color of the lines may also be adjusted by clicking inside the line field and holding the left click on the mouse The same styles are applied to both the signal and current traces Lane 1 iph EU Turn line Lane 2 Iph visualization on off Lane 3 Iph Lane 4 Iph Lane 5 Iph Hold left click on mouse to change line color or style 21212
72. the well 3 Repeat step 2 for remaining wells BluePippin Operations Manual software v 6 13 cassette definition Set 15 11 2 12 Graph Display Image Display 888 744 2244 www sagescience com support sagescience com Running a Protocol The default screen on the BluePippin is a tabbed format The Main Tab is the first tab and the default screen 12 1 Overview The Main Tab is the screen with which users load protocols run the instrument and monitor optical signal and electrophoresis Main Tab Protocol Editor f Log Review f File Manager System Options Sample ID Current Elution Timer Reference Idle Separate Elute 4 0 00 00 00 00 1 Al i zl 0 00 00 00 00 1 Al p OO Lo oc ag O TS 000 cco nn 1 1 00 00 00 15 00 30 00 45 01 00 01 15 01 30 01 45 02 00 02 15 02 30 Time hh mm Protocol Name protocol xyz Progress hh mm ss Voltage Y CI ock 99 9 07 Feb 2012 17 07 31 Remote Access Estimated Completion Time 07 Feb 2012 18 06 49 Blue Pippin 12 2 Starting a Run 1 Gotothe Main Tab 2 Place the calibration fixture on the optical nest See Section 5 for the detailed information on the calibration procedure 3 Press CALIBRATE A calibration window will launch Lane Status Panel Protocol Status Panel Control Panel 4 In the Target ph mA field enter 0 60 for dye free cassettes or 0 30 fo
73. tion and end is also based on the factory calibration settings in the cassette definition file and the user input size range settings in a run protocol Section12 BluePippin Operations Manual software v 6 13 cassette definition Set 15 1 2 888 744 2244 www sagescience com support sagescience com Safety and Precautions Icons Used In this manual the following icons will be used to provide the user with information pertinent to the use of the BluePippin A A Caution Warns the user that injury or instrument damage may occur if the contents of the warning are not properly followed High Voltage Warns of the risk of electrical shock if the contents of the warning are not properly followed Important Provide important information about the proper use of the system that may influence the quality of the result Information Provides additional information regarding the function of the system or applications for which is used Safe Use Guidelines The BluePippin system is designed to operate under the following environmental conditions Pollution Degree 2 Installation category 2 Altitude 2000m Indoor use Ambient temperature 17 32 C Humidity 10 80 non condensing Standard laboratory precautions should be taken when handling BluePippin Gel cassettes and operating the BluePippin Wear a lab coat safety glasses and gloves Use in proximity of an eye wash station and or running water BluePippi
74. tional sample Important A pause will require the user to manually resume the protocol from the Main Screen controller The instrument lid may be opened during the pause and sample retrieved 6 Enter Sample ID information Important Minimum size ranges are may not be the ideal for some applications Very narrow size ranges collect less DNA than larger ranges Users should consider the requirement for distribution vs yield see sec 11 4 7 Save After programming a new protocol or editing an existing one the protocol file must be saved prior to use If the protocol is new press Save If a protocol has been edited a yellow alert will be displayed in the Protocol Changes Not Applied field Save will save the file under the previously saved name and Save As will allow a new name for the file to be applied Protocol Changes NOT Applied SAVE AS All protocol files are saved in a directory nome pippin BluePippin Protocols and may be accessed in the File Manager tab BluePippin Operations Manual software v 6 13 cassette definition Set 15 1 2 888 744 2244 www sagescience com support sagescience com 7 2 Range Mode programming broad size range collections The Range programming mode is a method for selecting DNA size range collections by a starting and ending base pair value in software The system will collect the fragment range between the start and end base pair values This option shoul
75. ttes store at R T o 1 package of adhesive tape for sealing elution wells o 1 reagent kit for 10 cassettes store at 4 C 40 ml of spare running buffer 440 ul DNA size markers 1 x 40 ul load cassette 500 ul loading solution 2X 800 ul 0 1 Tween20 in running buffer 2X 1 1ml Midori Green DNA binding dye 40 ul buffer chamber Tween solution is used to improve DNA recovery of larger DNA fragments BluePippin Operations Manual software v 6 13 cassette definition Set 15 3 5 888 744 2244 www sagescience com support sagescience com Important F uorescein labels will degrade at room temperature minimize time at RT for M labeled standards and markers Storage Conditions for Cassettes and Reagents The following storage requirements should be observed e Gel Cassettes Store at room temperature in the foil bags until ready for use e DNA Markers unlabeled store at 4 C e DNA Internal standards and markers labeled store at 4 C minimimize time at room temperature e Electrophoresis buffer may be stored at room temperature or 4 C e Loading solution store at 4 C Bring to room temperature before use e Tween Solution store at 4 C e Midori Green DNA binding dye store at 4 C BluePippin Operations Manual software v 6 13 cassette definition Set 15 3 6 888 744 2244 www sagescience com support sagescience com Achieving Best Results from the BluePippin The BlueP
76. u for purchasing the BluePippin from Sage Science The BluePippin is an automated preparative gel electrophoresis system We urge you to read this manual to familiarize yourself with the system s capabilities and precautions System Overview There are three components to the BluePippin system Instrument The instrument contains an epifluorescence detector an electrophoresis power supply an electrode array and a Linux based single board computer The system computer is accessed by an external LCD monitor mouse and keyboard The electrode array is located in the top of the sliding instrument cover and is not user accessible Software System software allows the user to enter parameters that define the DNA fragment size ranges or DNA bands that are to be collected The software also logs fluorescence and electrical current data which may be analyzed in a review screen Gel Cassettes Pre cast disposable agarose cassettes are manufactured by Sage Science Cassettes are supplied in kits that contain 10 cassettes and sufficient reagents to complete all collections Kits have been developed to address DNA size range collections at various size ranges or DNA band capture Please refer to Section 21 for kit descriptions and ordering numbers Collection Strategies Cassette kits employ the following strategies For Size Selection of smaller DNA fragments 90 bp 1 5 kb Dye free gels with labeled internal standards Fl
77. un times up to 7 hours for low voltage runs The Run Time Selector consists of a field and a check box with the following default settings Run Time hh mm End Run when Elution is Completed x The 8 hour default value in the Run Time field ensures that a run is not unintentionally ended early The checked box in the End Run when Elution is Completed field will automatically end a run when every lane that has a programmed elution has completed its collection Users may adjust the desired run time by referring to Section 21 where estimated run times are listed for each cassette definition Users should be aware of the following when adjusting the run time parameters e farun time is entered based on run time estimates from Section 21 make sure adequate time is added to the estimate as a safety e The Run Time field will take precedence When the run time is achieved the run will end regardless of whether the elutions are complete To end a run manually go to the Main Tab and press STOP Completed elutions may be verified in the Lane Status Panel if there is a value is in the Elution Timer field and the Separate button is lit BluePippin Operations Manual software v 6 13 cassette definition Set 15 6 5 888 744 2244 www sagescience com support sagescience com 6 5 Assigning a Reference Marker external marker 1 Determine the lane into which the reference marker will be loaded 2 I
78. uorescein labeled DNA internal standards are added to samples when run The rates of migration of the internal standards are used to determine the collection timing within the sample lane Up to five samples may be run per cassette The DNA sequence of internal standards may be found at www sagesscience com support BluePippin Operations Manual software v 6 13 cassette definition Set 15 1 1 888 744 2244 www sagescience com support sagescience com For Size Selection of large DNA fragments gt 2kb Dye free gels with labeled external marker A fluorescein labeled DNA marker set is loaded in a dedicated sample lane during a run The rates of migration of the markers are used to determine the collection timing the remaining sample lanes Four samples and one DNA marker must be run per cassette For DNA Band Capture gt 2 kb DNA binding dyes with external standards A DNA marker set is loaded in a dedicated sample lane during a run A DNA binding dye Midori Green is loaded into the buffer chambers of the separation channels of all sample lanes During electrophoresis the DNA binding dye enters the gel and provides fluorescent detection of the DNA bands and markers The rates of migration of the markers are used to determine the band capture thresholds in the remaining sample lanes Four samples and one DNA marker must be run per cassette DNA binding dye must also be added by the user Collection timing start dura
79. w window Extract All Fa Scan for Viruses Fa Move to Quarantine Open with Ei Adobe Drive CS4 bP Send to k Cut Copy Create shortcut Delete Rename BluePippin Operations Manual software v 6 13 cassette definition Set 15 20 1 888 744 2244 www sagescience com support sagescience com 5 A window which will launch to prompt you to select a destination where to extract files The prompt will suggestcreating folder based on the zip file name Delete the destination folder name so that only the root directory appears E in the example below w 4 Extract Compressed Zipped Folders Ww 4 Extract Compressed Zipped Folders Select a Destination and Extract Files Nn Select a Destination and Extract Files Files will be extracted to this folder Files will be extracted to this folder E pip pinup grade463 Show extracted files when complete V Show extracted files when complete CE es Correct Files will be extracted to this folder Not Correct Files will be extracted to this folder 6 The final USB file structure should be as shown The zip file may be deleted a Removable Disk E PippinUpgrade J Bin di Cassettes gt Upgrade pippinupgrade463 BluePippin Operations Manual software v 6 13 cassette definition Set 15 20 2 888 744 2244 www sagescience com support sagescience com 20 2 Upgrading the Pippin Instrume
80. when it is in position to be eluted See cassette specifications at the end of this manual to determine the best timing for manual runs BluePippin Operations Manual software v 6 13 cassette definition Set 15 17 2 888 744 2244 www sagescience com support sagescience com 18 Protocol Name Protocol Parameters Optical Signal Analyzing Runs Log Review Tab The default screen on the BluePippin is a tabbed format The Log Review is the third tab Overview The Log review screen is used to review data and programming parameters from previous size selection runs All programming parameters are displayed as originally entered in the Protocol Editor Optical and electrical current data from the run are displayed in graphical windows Specific regions of either window can be selected and expanded as needed for detailed analyses These data are useful for refining size selection performance and verifying instrument performance Log Review Tab Log file Directory Cassette Type DNA Marker Times and Values Current Trace BluePippin Operations Manual software v 6 13 cassette definition Set 15 18 1 888 44 2244 www sagescience com support sagescience com Opening a Log file in the Review Screen Note A stand alone Review Log Program Is available for PCs Contact sage science support i Click the Log Review tab along top of the screen li To select a log file press the folder icon in next to

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