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Blue Pippin User Manual - UTA

1. Buffer an m L gt Buffer Chamber k g er 2 Legume Chambers DR an E f _ If Check behind elution modules for bubbles Tilt cassette to clear 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 fully 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 w
2. sage science Blue P DPI n DNA Size Selection System Operations Manual Software v 6 00 Cassette Definition Set 10 978 744 2244 www sagescience com e support sagescience com Sage Science Inc Suite 3150 500 Cummings Center Beverly MA 01915 2013 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 00 cassette definition Set 10 I 978 744 2244 www sagescience com e support sagescience com TABLE OF CONTENTS 1 INTRODUCTION oa eee tec ccs sain E nan canne Ga es mue 1 1 2 SAFETY AND PR CAUTIONS cscccnsssastonseceeesesvese case onsescenssetenacstsconsansuconseeneceseennntenstans 2 1 3 UNPACKING AND INSTALLATION ccceteceeeeeeseeeeeeseeeeenseeeeeneeseenseeeeeneeseesseesoenseneoens 3 1 4 ACHIEVING BEST RESULTS FROM THE BLUEPIPPIN ccccsssseeeeseseeeeenseeeeenees 4 1 5 SAMPLE PREPARATION nan snsnsssaen ns mass mens adm anne ansence tes scescts 5 1 6 PROTOCOL PROGRAMMING GUIDE cccteccceeeeeeeeeeeeeeeeeeneeeeaeeeeeneeeeaseseeneeeeneeaeanenes 6 1 6T OVE WCW ascites cece ecw ene eeee use remcwunt aa 6 1 62 Programming UIA REY ssc site ee semen signetiners oe tise cic saietnsaaie aa a nain 6 2 6 3 Selecting a Cassette Definition cccccceeeesssssssssssssseeenseesseeeeeeeeeeeeeseoooooeeeeesssnseeseseeeesses
3. 6 5 Assigning a Reference Marker external marker 1 Determine the lane into which the reference marker will be loaded 2 Inthe 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 a LEIE ENCE LATIE been loaded HS QU reference off of marker lane USE INTERNAL STANDARDS 7 APPLY REFERENCE TO ALL LANES Remaining lanes will 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 00 cassette definition Set 10 6 6 978 744 2244 www sagescience com e 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 INTERNA
4. 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 collections of large DNA fragments especially with pulsed field protocols can also appear counter intuitive with respect to timing Rate of Migration Separation 25 Faster Figure 4 3 An illustration comparing the relative rate of electrophoresis in the separation and elution channels BluePippin Operations Manual software v 6 00 cassette definition Set 10 4 2 9 978 744 2244 www sagescience com e support sagescience com Sample Preparation Input Sample Characteristics When running the BluePippin characteristics of inout 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
5. Files will be extracted to this folder 6 The final USB file structure should be as shown The zip file may be deleted Removable Disk E di PippinUpgrade Bin di Cassettes di Upgrade pippinupgrade465 BluePippin Operations Manual software v 6 00 cassette definition Set 10 19 2 978 744 2244 www sagescience com e support sagescience com 19 2 Upgrading the Pippin Instrument 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 SMAPSHOT IMFO SHUTOO TH 3 From the Information Window press SOFTWARE UPGRADE Blue Pippin 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 00 cassette definition Set 10 19 3 978 744 2244 www sagescience com e support sagescience com 5 Press OK at the warning prompt the Software Upgrade Window will launch 6 Press INSTALL UPGRADE this will take about 5 seconds INSTALL UPGRADE UNMOUNT 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 retu
6. Warnings Some elution ranges will cause chamber overflow Some start values missing Some end values missing some start fend values ill defined some BPstart or BPthresh values out of ladder range BluePippin Operations Manual software v 6 00 cassette definition Set 10 6 9 978 744 2244 www sagescience com e support sagescience com 7 Programming Size Selections Tight Range and Timed Modes 7 2 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 e
7. 6 00 cassette definition Set 10 13 2 978 744 2244 www sagescience com e support sagescience com 13 3 Improving Product Recovery with the Field Reversal Providing a brief current reversal at the end of a sample elution can improve sample recovery by up to 20 This option is set in the System Options Tab of the Pippin Software The use of this option is recommended Figure 10 2 below shows the improvement that can be achieved Figure 10 3 shows the setting option in software The default setting is ON When activated the current will be reversed on all subsequent runs until the user changes the setting through the System Options tab 2 cassette pBR 322 Msp digest 40 min elution With current reversal b gt Pots Without current reversa kd 200 300 400 Size of Fragment Bp Figure 13 2 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 Name POO LI D F 5 Ewer F o tt Use Reverse Field for Sample Recovery Press button and then press Accept BluePippin Operations Manual software v 6 00 cassette definition Set 10 13 3 978 744 2244 www sagescience com e support sagescience com Figure 13 3 System Options Tab with expanded view of Reverse Field Option 13 4 Improving Product Yield by Selecting a Wider Size Range The BluePippin can produce very narrow size distribution
8. BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 10 978 744 2244 www sagescience com e support sagescience com 23 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 l l l l l l 01 20 01 40 0200 02 20 02 40 03 00 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 11 978 744 2244 www sagescience com e support sagescience com 23 11 0 75 Agarose Dye Free 10 18 kb Marker U1 Pulsed Field external marker Use Product Number BMF7510 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 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 39 Run times are affected by temperature an
9. 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 automatically 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 00 cassette definition Set 10 18 2 978 744 2244 www sagescience com e support sagescience com Protocol Files The protocol file directory is accessed by pressing the GO TO PROTOCOLS button Protocol files have a pppro
10. 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 O3 00 O4 00 06 00 Time Ahram 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 9 978 744 2244 www sagescience com e support sagescience com 23 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 01 00 01 30 02 00 02 30 Time hh mm Estimated Collection Times for Minimum Size Range Tight Targets bp hr min D 000 1 51 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 23 1 for specification definitions
11. Tight and Modes Indicators ae ee samen drapes parameters P parameters Loft Loft Ka 6 2 Programming Summary The following is a summary of the steps required 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 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 00 cassette definition Set 10 6 2 978 744 2244 www sagescience com e 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 Ei 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 t
12. com e 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 00 cassette definition Set 10 12 5 978 744 2244 www sagescience com e 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 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
13. 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 the selected DNA Sample 40 ul Bufer OR Sample 40 ul Figure 11 1 An illustration of the electrophoretic effect on a DNA sample when a sample well in not completely filled BluePippin Operations Manual software v 6 00 cassette definition Set 10 11 1 978 744 2244 www sagescience com e 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
14. mobility and reduce recovery Specifications subject to change without notice BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 1 978 744 2244 www sagescience com e support sagescience com 23 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 0 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 2 978 744 2244 www sagescience com e support sagescience com 23 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 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 Dis
15. 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 Section 13 4 for details Collection ranges 10 5 CV 6 6 CV Input gDNA 5 ug 5 0 CV Tight Figure 4 1 comparison of the relative sample yield between tight and broader range size selections of DNA BluePippin Operations Manual software v 6 00 cassette definition Set 10 4 1 978 744 2244 www sagescience com e 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 ii Figure 4 2 An illustration of the time and base pair difference between the detector and branch point
16. 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 Editar 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 a 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 00 cassette definition Set 10 12 6 978 744 2244 www sagescience com e support sagescience com 13 Sample Collection Sample Well Elution Module Buffer al A Buffer Chamber A gt Chambers 13 1 Overview 1 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 A 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 us
17. used as an approximate guideline Performance Specifications 100 600 bp Minimum Size Distribution as CV Sample Recovery 50 80 Accuracy lt 5 Reproducibility lt 5 Specifications subject to change without notice see page 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 5 978 744 2244 www sagescience com e support sagescience com 23 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 1200 00 20 00 25 56 Time hh mm 1500 RE 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 6 978 744 2244 www sagescience com e support sagescience com 23 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 Typi
18. 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 the well 3 Repeat step 2 for remaining wells BluePippin Operations Manual software v 6 00 cassette definition Set 10 11 2 978 744 2244 www sagescience com e support sagescience com 12 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 Lane each Status rap Panel Display ae 1 j Va ce M pp m ton ee p fecell LOS ee cele LE Protocol ec ov os aa a So na uw rt ox Status mage Panel Display Control Panel Blue Pippin ee ou ose aa ne HE SE ai Ow du ox EP ER ES 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
19. 2 16 978 744 2244 www sagescience com e 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 00 cassette definition Set 10 16 1 978 744 2244 www sagescience com e support sagescience com 4 Press Pause Resume and Stop buttons are also operative as in automated runs TESI START CALIBRATE SNAPSHOT INFO SHUT ENT Note There is a delay between the time that a fragment is detected and visible on the graph or D image and 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 00 cassette definition Set 10 16 2 978 744 2244 www sagescience com e support sagescience com 17 Prot
20. 3 Press CALIBRATE A calibration window will launch 4 Inthe Target ph mA field enter 0 60 for dye free cassettes or 0 30 for Midori Green dye cassettes BluePippin Operations Manual software v 6 00 cassette definition Set 10 12 1 978 744 2244 www sagescience com e support sagescience com Enter 0 60 for dye free runs Enter 0 30 for Midori Green runs LED Calibration Target ph 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 Clock 07 Feb 2012 170731 r bP at A oo Ta s 07 Feb 2012 1808 49 8 Load a 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 mo SHUTDOWN BluePippin Operations Manual software v 6 00 cassette definition Set 10 12 2 978 744 2244 www sagescience com e support sagescience com 10 An LED Calibration window will launch If the instrument has not be
21. ING SAMPLES anne anses anne ane anses 11 1 12 RUNNING A PROTOCOL nn se sms aomocm sas mens cs amnennnsncsnesnsn encens 12 1 AN SENG a een a ed eee eee eee 12 1 22 Saung NE LU 1 PR RE Re TE 12 1 TS Montong a RON PP RS EE Re 12 4 124 VOC FIGS ae nes ns ne 2 en ananas ane sn ocean once 12 6 13 SAMPLE COLLECTIONS dete tates dee ara ies clece deteea tice eae cs 13 1 Wed OV GE WIC N E tect emcenn arate ci E E E A A ca A aha A E A 13 1 13 2 Intrinsic Sample Recovery on the BluePippin ss sssnnnnnnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnmnnn 13 2 13 3 Improving Product Recovery with the Field Reversal ss 13 3 13 4 Improving Product Yield by Selecting a Wider Size Range 13 4 TA SYSTEM VALIDATION Sas nrc san eee te 14 1 19 SYOTEM OPTIONS TAB sens ue 15 1 16 RUNNING IN MANUAL MODE nn nnsnnrrermnaneennnnnnennnnnennnnnnnennenennnnneeennneennnnnnee 16 1 17 ANALYZING RUNS LOG REVIEW TAB ccccceeseeeteneeeeseeeeeneeeeneeeeaseeeeneeeeaeeeneneenens 17 1 18 MANAGING FILES FILE MANAGER TAB ccccceeseeeeseneeseeneeeeeeneeseenseeeeeneeseeenees 18 1 Tor ENG ira ta eg a eg a E eee 18 1 oe RS TYPES atria cect cece ee 18 2 19 UPGRADING SOFTWARE ae ans ececsunvoneches vorenbexuseeuibew are 19 1 19 1 Extracting the Files to a USB flash drive cccccessssssseeeeeeesneeeeseosneeeeseoesseeeseeoenseeessoeenneees 19 1 19 2 Upgrading the Pippin Instrument Softwar e ccccssssseeeseeessseeeeeeess
22. L STANDARDS internally loaded standard APPLY REFERENCE TO ALL LANES Tight Range Time Peak Ref Lane Figure 6 2 The correct configuration for a protocol 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 00 cassette definition Set 10 6 7 978 744 2244 www sagescience com e 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 00 cassette definition Set 10 6 8 978 744 2244 www sagescience com e support sagescience com Warnings Text Box lf there are errors or exceptions within a programmed protocol a message will appear in the Warnings text box identifying the nature of the discrepancy
23. NA run on a 2 0 Agarose Cassette Important Data are not intended to imply guaranteed results or performance Control 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 00 cassette definition Set 10 14 2 978 744 2244 www sagescience com e support sagescience com 15 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 Date and Time 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 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 Ti
24. 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 00 cassette definition Set 10 10 1 978 744 2244 www sagescience com e support sagescience com 4 Inspect bottom of cassette for bubbles in the detection region of the gel columns lf a cassette has been jarred during shipping the agarose column can delaminate from the plastic bottom of the separation channel forming a thin 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 be
25. 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 should 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 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
26. 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 size 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 aN RE 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 wg 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 00 cassette definition Set 10 5 1 978 744 2244 www sagescience com e support sagescience com 6 Protocol Programming Guide The default screen on
27. aseline before each run O O Or Or OO Figure 3 1 Adapter plug for non US power cord installation BluePippin Operations Manual software v 6 00 cassette definition Set 10 3 1 978 744 2244 www sagescience com e 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 lbs 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 all
28. b Marker S1 Pulsed Field external marker 23 10 23 10 0 75 Agarose Dye Free 5 10 kb Marker S1 Pulsed Field external marker 23 11 23 11 0 75 Agarose Dye Free 10 18 kb Marker U1 Pulsed Field external marker 23 12 23 12 0 75 Agarose Dye Free 18 27 kb Marker T1 Pulsed Field external marker 23 13 23 13 0 75 Agarose High Range Targets Marker W1 Pulsed Field ext mrk 40 amp 50kb 23 14 BluePippin Operations Manual software v 6 00 cassette definition Set 10 978 744 2244 www sagescience com e support sagescience com Introduction Thank you 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 curre
29. cal times to detector 00 30 00 40 00 50 01 00 0110 01 20 Time hh mm Estimated Collection Times for DNA bands 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 978 744 2244 www sagescience com e support sagescience com 23 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 30 02 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 S CE CC I 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 8 978 744 2244 www sagescience com e support sagescience com 23 8 0 75 Agarose Dye Free 1 6 kb Marker
30. d 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 12 978 744 2244 www sagescience com e support sagescience com 23 12 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 Time to Time to Collect bp hr min 18 000 3 45 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 13 978 744 2244 www sagescience com e support sagescience com 23 13 0 75 Agarose High Range Targets Marker W1 Pulsed Field external marker 40 and 50kb Use Product Number BHG7510 Use Cassette Definitions 0 75 A
31. de 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 19 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 7J 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 7J 4 Right click on the zip file and select Extract All Marne Date mod TE pippinupgre da Open Open in new window Extract All Fa Scan for Viruses Fa Mowe to Quarantine Open with H Adobe Drive CS4 bP Send to k Cut Copy Create shortcut Delete Rename BluePippin Operations Manual software v 6 00 cassette definition Set 10 19 1 978 744 2244 www sagescience com e 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 Correct Files will be extracted to this folder Not Correct
32. 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 00 cassette definition Set 10 10 6 11 978 744 2244 www sagescience com e support sagescience com Loading 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
33. e 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 target 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 00 cassette definition Set 10 8 1 978 744 2244 www sagescience com e support sagescience com BP Thresh Start Exp End Exp Sample ID Template 0 90 C 0 90 E EOE EEN ENE 000 090 0 E 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 Ex
34. e 3 Lane 2 Lane 1 hya Iteration LED Current counts Photocurrent mA re 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 00 cassette definition Set 10 9 3 978 744 2244 www sagescience com e support sagescience com 7 The LED photocurrents will automatically adjust to a factory setting and the Calibration Status field will contain the message Calibration OK 8 Press EXIT to return to the Main Tab BluePippin Operations Manual software v 6 00 cassette definition Set 10 9 4 978 744 2244 www sagescience com e 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 Inspect the 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
35. e v 6 00 cassette definition Set 10 1 5 978 744 2244 www sagescience com e support sagescience com 8 Programming DNA Band Capture with Midori Green 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 th
36. eeeeseeessseeseeoenseeessoeenseees 19 3 20 WARRANTY AND SERVICE cccecsceseeseeeeeeeeeeeeneeeeeeneeseenseeseeaeeseeaseeseeaseeeneseesoanenses 20 1 21 ORDERING INFORMATION ccccecsceeeseeeeesseeeeeneeeeeneeseeaseeeeeseeeeaneesoeaseseneseesoenseeses 21 1 22 INSTRUMENT SPECIFICATIONS cccesccesesseeeseseeesenseeeeenseeseenseeeeaseesoenseeeeeseesoeaseesos 22 1 BluePippin Operations Manual software v 6 00 cassette definition Set 10 978 744 2244 www sagescience com e support sagescience com TABLE OF CONTENTS CONT D 23 GEL CASSETTES SPECIFICATIONS 0 0 1 cccceeecsneseneeeneeeneeeneeenesenesenssenseenesenesoneees 23 1 23 1 3 Agarose Dye Free 90 bp 250 bp Marker Q2 internal standards 0008 23 2 23 2 2 Agarose Dye Free 100 bp 600 bp Marker V1 internal standards 23 3 23 3 2 Agarose Dye Free 100 bp 600 bp Marker X1 int stds 25 ul elution well 23 4 23 4 2 Agarose Dye Free 100 bp 600 bp Marker M1 external marker 23 5 23 5 1 5 Agarose Dye Free 250 bp 1 5 kb Marker R2 internal standards 23 6 23 6 1 5 Agarose Band Capture 250 1500 bp Marker A1 external marker 23 7 23 7 0 75 Agarose Band Capture 2 10 kb Marker D1 Pulsed Field external marker 23 8 23 8 0 75 Agarose Dye Free 1 6 kb Marker S1 Low Voltage external marker 23 9 23 9 0 75 Agarose Dye Free 1 6 k
37. elow Revert to default view Grab and move 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 BluePippin Operati ANALYZE PEAKS SMAFSHOT Set 10 17 5 978 744 2244 www sagescience com e support sagescience com 18 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 the left anda target directory portable flash media i e USB key on the right File Manager Tab Pippin Destination File Drive File Directory Directory Pippin Destination Files Drive Files File Types File Commands BluePippin Operations Manual software v 6 00 cassette definition Set 10 18 1 978 744 2244 www sagescience com e support sagescience com 18 2 File Types GO TO LOGS GO TO 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
38. en calibrated or if it has been calibrated 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 cassetie chose jid sag CALIBRATE Load WAT To start wibaut coltirahon lead TANT cassette and START To rancet ralbedion and profocal 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 00 cassette definition Set 10 12 3 978 744 2244 www sagescience com e 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 Sample D Reference e Green Separation If a lane is separating fragments the Separate indicator will be green e Orange Elution Ifa lane is eluting a size range the Elute indicator will be orange and the elution timer will be active 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 00 cassette definition Set 10 12 4 978 744 2244 www sagescience
39. eneanononoes 6 4 CS RO MS cs ec E ce ec a eee eee cee eee ee ccete mete ee ceeec 6 5 6 5 Assigning a Reference Marker external marker cccccseesseeeeeeeeeeeeeeeneseeeeeeeenseeeeseeenseeeeseneaes 6 6 6 6 Assigning Reference Markers internal standards sense 6 7 67 WArnINgS and INGICAIONS 5 sss simnn snnas ste cases names sntee es 6 8 7 PROGRAMMING SIZE SELECTIONS TIGHT RANGE AND TIMED MODES 7 1 7 2 Tight Mode programming minimum size range collections sens 7 1 7 2 Range Mode programming broad size range collections ns 7 3 7 9 Time Mode programming timed collections ccccceceeeeeseeeseeeenseeeseoeenseeeeenenseeeeeeoenseeessooenes 7 4 8 PROGRAMMING DNA BAND CAPTURE WITH MIDORI GREEN PEAK MODE 8 1 9 OPTICAL CALIBRATION csisccsstesccsseneseccsacesasasencsarcceccinccssacsentsanesceasnccsartecsesencscaciwecssaecesces 9 1 10 PREPARING A CASSETTE senc snne ee 10 1 10 1 Visually Inspect the Cassette cccceeesssseessseeeeeeeeeeeesnseeeeeeseneeeaaaeeeeeeeeneeaeaaaeeeeesenooesseseneesaoons 10 1 10 2 Prepare the Cassette for Loading sm nnsssssnenernnssnnnenneenennnssnneneeeeeennnnns 10 3 103 CONTINUE Tes a ee 10 4 10 4 Adding Midori Green for Band Capture Cassettes ns 10 6 BluePippin Operations Manual software v 6 00 cassette definition Set 10 978 744 2244 www sagescience com e support sagescience com TABLE OF CONTENTS cont d ML COAD
40. garose Dye Free 0 75 DF 40 kb Marker W1 tight 0 75 Agarose Dye Free 0 75 DF 40 kb Marker W1 broad 0 75 Agarose Dye Free 0 75 DF 50 kb Marker W1 tight 0 75 Agarose Dye Free 0 75 DF 40 kb Marker W1 broad DNA Marker W1 Typical times to detector l l l 02 30 05 00 0ra Time hh mm Estimated Collection Times nen ent to 40kb tight oe oleae time to size select from one sample 50kb tight add time to each additional sample lane size selection 50kb broad 40kb tight 40kb broad 50kbtight 50kb broad Minimum Size gt 5 J Reproducibility Specifications subject to change without notice see page 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 14
41. gt 2 kb DNA binding dyes with external standards A DNA marker set is loaded in a dedicated sample lane during arun 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 duration 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 Section1 2 BluePippin Operations Manual software v 6 00 cassette definition Set 10 1 2 978 744 2244 www sagescience com e 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 A D 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
42. he 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 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 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 00 cassette definition Set 10 7 4 978 744 2244 www sagescience com e 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 softwar
43. ill 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 MOT 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 9 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 Enter a 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 If 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 t
44. in na uw ein ox Protocol Status Image Panel Display Control GA Panel ee ni wa aa we an he we oe dit ox hee forme UM RENTE MNT Ce TTEN 1 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 2 Press CALIBRATE on the Control Panel TEST START CALIBRAT SMAPSHOT IMFO SHUTDOWN i BluePippin Operations Manual software v 6 00 cassette definition Set 10 9 1 978 744 2244 www sagescience com e 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 software v 6 00 cassette definition Set 10 9 2 978 744 2244 www sagescience com e 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 LEO Calibration Target ph mA La ne 5 as Lane 4 Lan
45. ing the empty rinse cassette supplied with the instrument 2 Samples from standard cassettes without sealed elution modules will be recovered in 40 65 ul of electrophoresis buffer depending on the length of the elution step Samples from cassettes with tape sealed elution modules will be recovered in a fixed volume of 40 ul 3 When all samples are collected remove used cassette from instrument and dispose of it properly 4 Additional DNA product can be washed from the elution modules with a nonionic detergent After the initial product is removed and saved add 40 ul of electrophoresis or TE 0 1 Tween 20 to the elution module After a brief incubation 1 min remove the buffer Tween solution and combine it with the initial product Tween wash is especially important for achieving a good recovery of samples that have not been deproteinized 5 For collections of larger fragments DNA gt 2kb DNA recovery from the BluePippin may be improved by up to 100 Tween solution is supplied by Sage Science in the reagent BluePippin Operations Manual software v 6 00 cassette definition Set 10 13 1 978 744 2244 www sagescience com e support sagescience com 9 packs for cassette kits that collect over 2 kb Collections lt 1kb the improvement in recoveries are between 10 20 with Tween solution Important Eluted samples should not be left in the cassette for longer than a couple of hours to avoid poor recovery due to adsorps
46. ion 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 samples can be used directly for ligation and amplification without buffer exchange 13 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 Figure 10 1 below 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 Le Inn i i 1 1 f 1 Bioanalyzer Standard lt lt Figure 13 1 An example of the intrinsic recovery of 5 kb band on a 0 75 agarose cassette BluePippin Operations Manual software v
47. it 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 D I x TEE FE Rite TE 0000 0015 0030 0045 0100 0115 0130 0145 0200 0215 0230 si BPENd P avss 40 Figure 14 1 Control DNA profile with reference marker and DNA extraction BluePippin Operations Manual software v 6 00 cassette definition Set 10 14 1 978 744 2244 www sagescience com e support sagescience com protocol for a 2 agarose DNA control kit run 60 Target 150 bp 40 Ave Size 149 bp Conc ng ul 3 60 2U g f ee EER JRI a 15 50 100 200 300 400 500 700 1500 Figure 14 2 Example of a Bioanalyzer result for the 150 bp target of a Control D
48. ith the cassette package or pipette from the buffer chambers BluePippin Operations Manual software v 6 00 cassette definition Set 10 10 3 978 744 2244 www sagescience com e 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 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 chan
49. low 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 Sample Well Ehution Well i Buffer oe Buller Chamber G gt eens g RER AR ES 7 Optical Detection eg Region Figure 10 3 The region of optical detection BluePippin Operations Manual software v 6 00 cassette definition Set 10 10 2 978 744 2244 www sagescience com e 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
50. lution 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 Sie a Test parameters gt Elution Channels FAIL replace buffer and retest If it fails again do not use for collections Separation Channels FAIL do not use lane BluePippin Operations Manual software v 6 00 cassette definition Set 10 10 5 978 744 2244 www sagescience com e support sagescience com Figure 10 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 add Midori Green Dye r Ser y Tr l Li LA Se Sample Well Elution Module x Buffer Chambers Buffer Chambers Optica 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
51. me 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 00 cassette definition Set 10 15 1 978 744 2244 www sagescience com e 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 M 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 setting Instrument Name Blue 2 IOF 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 BluePip 15
52. nel 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 CAT BluePippin Operations Manual software v 6 00 cassette definition Set 10 10 4 978 744 2244 www sagescience com e 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 eT mt Conus les 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 e
53. nt 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 Fluorescein 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 00 cassette definition Set 10 1 1 978 744 2244 www sagescience com e 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
54. nter the lane into which the marker will be loaded into the Reference Lane field 1 is the default value li press the APPLY REFERENCE TO ALL 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 00 cassette definition Set 10 1 1 978 744 2244 www sagescience com e support sagescience com 5 Optional Enter a BP pause value Ifa 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 elec
55. o be collected using starting and ending base pair values e Time allows users to program extractions using the starting and 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 D Note Programming modes may be independently applied to individual sample lanes 6 Enter Sample ID or description optional BluePippin Operations Manual software v 6 00 cassette definition Set 10 6 3 978 744 2244 www sagescience com e 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 0 0 75 DF 2 6kb Marker 51 0 70 DF 3 10kb Marker 51 Ofo DF Low Voltage 1 6kb Marker 31 EAP AND ALL COLLAPSE ALL CANCEL BluePippin Operations Manual software v 6 00 cassette definition Se
56. ocol 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 i Tab Log file Directory Cassette Type as ta nT DNA a Marker Times and Values an Erer Current Trace BluePippin Operations Manual software v 6 00 cassette definition Set 10 17 1 978 744 2244 www sagescience com e 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 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 00 cassette definition Set 10 17 2 978 744 2244 www
57. ow 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 00 cassette definition Set 10 3 2 978 744 2244 e WWww sagescience com e support sagescience com USB Ports 4 Power Switch Figure 3 2 Rear Panel Ports VGA monitor port Power Entry Port Key board Monitor Cable Power Cable Figure 3 3 Rear Panel Connections BluePippin Operations Manual software v 6 00 cassette definition Set 10 3 3 978 744 2244 www sagescience com e 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 00 cassette definition Set 10 3 4 978 744 2244 www sagescience com e 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 sol
58. pansion factors values may be used to widen or narrow the collection criteria based on user needs BP Start BP End Start Exp A End Exp 0 90 r A 1 10 90 of BP Start value s 110 of BP Start value L gt LL D Y a L e e gt v n E r a v Figure 8 1 Expansion factors values widen the peak collection timing to ensure complete capture of the peak BluePippin Operations Manual software v 6 00 cassette definition Set 10 8 2 978 744 2244 www sagescience com e 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 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
59. 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 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 asample 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 00 cassette definition Set 10 1 3 978 744 2244 www sagescience com e 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 w
60. 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 BluePippin Operations Manual software v 6 00 cassette definition Set 10 2 1 978 744 2244 www sagescience com e 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 b
61. rn 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 Harme Pipl SV Version 5 30 IP Address 127 0 0 1 D Note The system should be ready to use re boot is not necessary BluePippin Operations Manual software v 6 00 cassette definition Set 10 19 4 978 744 2244 www sagescience com e support sagescience com 20 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 replacement 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 disas
62. s 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 V1 BDF2010 2 agarose dye free 100 600 bp 25 ul elution well X1 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 10 kb S1 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 W1 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 4Oul BluePippin Operations Manual software v 6 00 cassette definition Set 10 21 1 978 744 2244 www sagescience com e support sagescience com Ordering Information Cont d Control DNA for Performance Validation Control DNA for 3 Gel Cassette Int Std 90 250 bp 5 ug load 16 loads Control DNA 2 Gel Cassette Int Std 100 600 bp 5 ug load 16 loads Control DNA for 2 Gel Cassette Ext Mkr 100 600 bp 5 ug load 16 loads Control DNA for 1 5 Gel Cassette Int Std 250 1500 bp 2 ug load 16 loads Control DNA for 0 75 Gel Cassette Ext Mkr 1 10 kb 5
63. s 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 00 cassette definition Set 10 3 6 978 744 2244 www sagescience com e support sagescience com Achieving Best Results from the BluePippin The BluePippin 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 Successful Operation It is recommended that users closely follow the instructions outlined in the following sections Section 5 8 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 operation are Purity of the input sample See Section 5 Optical Calibration Section 9 Preparation of the cassettes Section 10 Sample loading See Section 11 4 2 Common Misconceptions Users should be aware of the following characteristics of the BluePippin System These are part
64. s 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 12 4 shows an example of the effect of size range selection vs yield Input gDNA 5 ug Aa pew lap val re ae i un Bioanalyzer Result Average End Size bp BP Start BP End Figure 13 4 Increasing yield by widening the size selection range D Information The per cent recovery specification is determined by collecting know amounts of i restriction fragments Section 10 2 Product yield from a sheared DNA sample is influenced by the fragment distribution and input profile of the sample BluePippin Operations Manual software v 6 00 cassette definition Set 10 13 4 14 978 744 2244 www sagescience com e 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 Agilent 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 K
65. sagescience com e support sagescience com 4 This will populate the Review Log Screen with data from the selected run log Protocol rr i Information ee 2 LS LA DNA Marker z Times and Values ace aCe am CE Electrophoretic T Current Traces a 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 FA Turn line Lane 2 Iph Gi visualization Lane 3 Iph FA on off Lane 4 Iph FA Lane 5 Iph fal Hold left click on Filtered Ref 1 etes aA line color or style Threshold FA BluePippin Operations Manual software v 6 00 cassette definition Set 10 17 3 978 744 2244 www sagescience com e 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
66. semble 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 disposed 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 00 cassette definition Set 10 20 1 978 744 2244 www sagescience com e support sagescience com 21 Ordering Information Catalogue No BluePippin Includes monitor keypad mouse and accessories BLUOOO1 Dye Free Gel Cassette
67. t 10 6 4 978 744 2244 www sagescience com e support sagescience com 6 4 Run Times Protocols can be run with widely variable run 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 Fi 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 00 cassette definition Set 10 6 5 978 744 2244 www sagescience com e support sagescience com
68. t 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 Casseite 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 functions are outlined below NE FLASH FOLDER UN MOUNT FLASH DRIVE Creates new Copies Copies folder on highlighted highlighted aed ae 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 Qata loss Failure to do so may cause data loss and problems with future upgrade procedures BluePippin Operations Manual software v 6 00 cassette definition Set 10 18 3 978 744 2244 www sagescience com e support sagescience com 19 Upgrading Software Pippin upgra
69. 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 range 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 Text Box File Commands BluePippin Operations Manual software v 6 00 cassette definition Set 10 6 1 978 744 2244 www sagescience com e 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 Programming Ref Lane
70. tion 470 nm excitation 525 nm emission Power Requirements 100 240 VAC 2 5 A 50 60 Hz Weight 15 lbs 7 kg Dimensions 7H X 11W X 21D in 18H X 28W X 53D cm Approvals CE CSA Country of Origin United States BluePippin Operations Manual software v 6 00 cassette definition Set 10 22 1 978 744 2244 www sagescience com e support sagescience com 23 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 Specifications Band Capture Cassettes Midori Green Minimum Sample Load 50 ng band Maximum Sample Load 2 ug band nae ae Sample Recovery 50 80 5 2X standard deviation of replicate 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
71. 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 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 00 cassette definition Set 10 8 3 978 744 2244 www sagescience com e support sagescience com 9 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 Lane Status Graph Panel Display oe of os e MN ae t
72. tribution as CV Sample Recovery 50 80 Specifications subject to change without notice see page 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 3 978 744 2244 www sagescience com e support sagescience com 23 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 23 1 for specification definitions BluePippin Operations Manual software v 6 00 cassette definition Set 10 23 4 978 744 2244 www sagescience com e support sagescience com 23 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 Marker 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 Run times are affected by temperature and broad range collections Times should be
73. trodes 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 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 00 cassette definition Set 10 1 2 978 744 2244 www sagescience com e 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
74. ug load 16 loads Control DNA for 0 75 Gel Cassette Ext Mkr 10 27 kb 5 ug load 16 loads Control DNA for 0 75 Band Capture Ext Mkr 1 kb fragment 5 ug load 4 loads Control DNA for 0 75 Band Capture Ext Mkr 3 kb fragment 5 ug load 4 loads Control DNA for 0 75 Gel Cassette Ext Mkr 10 kb fragment 5 ug load 4 loads Reagent Replacement Kits Dye Free with Internal Standards BDF3010 reagent replacement kit for 10 cassettes Marker Q2 BDF2010 reagent replacement kit for 10 cassettes Marker V1 BSF2010 reagent replacement kit for 10 cassettes Marker X1 BDF1510 reagent replacement kit for 10 cassettes Marker R2 Reagent Replacement Kits Dye Free with External Marker Reagent Replacement Kits Band Capture External Marker BLG7510 reagent replacement kit for 10 cassettes Marker D1 BLG1510 reagent replacement kit for 10 cassettes Marker A1 BluePippin Operations Manual software v 6 00 cassette definition Set 10 Catalogue No CIS3004 CIS2004 CON2004 CIS1504 CON7504 CON7604 CBC1501 CBC7501 CBC7601 Catalogue No RBF3010 RBF2010 RBF1510 Catalogue No RBE2010 RBQ2010 RLF7510 RUF7510 RMF7510 RHF7510 Catalogue No RGL7510 RGL1510 21 2 22 978 744 2244 www sagescience com e support sagescience com Instrument Specifications Specifications for BluePippin Instrument Electrophoresis Voltage 25V 100V 150V constant 100 V pulsed Optical detec
75. ution mix 10 ul sample 40 ml of spare 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 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 2X 800 ul 0 1 Tween20 in running buffer 2X1 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 00 cassette definition Set 10 3 5 978 744 2244 www sagescience com e support sagescience com Important Fluorescein 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 Marker
76. 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 im 0 82 0 70 0 60 r 050 T 5 5 040 Ci PL 0 08 l l l l l 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 RhimmssS 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 00 cassette definition Set 10 17 4 978 744 2244 www sagescience com e support sagescience com Current Graph 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 Lane Current mA CURRENT By 5 00 4 50 4 00 Salle 3 00 2 90 2 00 1 90 1 00 0 50 0 00 l 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 b

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