Pippin Pulse User Manual
Contents
1. 6 5 Pre set protocol example 4 S T50K WE 11 6 6 Pre set protocol example 5 B A20kb anoanoai iE 13 G EEGENT 14 8 0 Monitoring a Pippin Pulse Run 15 SLENGE ERUN E A E E E EEE 15 SPA E 15 9 0 Programming the Pippin Pulse ccc ccccsssscecceesssseeeceecsssseeeceecessseeceeesssaeeecesesssaeeeeeseeeaeeees 16 9 1 What is a Pippin GT He re e ele EE 16 GE EE AE GE 16 10 0 Software e TEE 19 WATE Man SOW EE 19 102 THC Edt O CO E 20 11 0 Pippin Pulse BlOg eeann a 21 sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 1 0 Introduction Thank you for purchasing the Pippin Pulse electrophoresis power supply The Pippin Pulse provides programmable pulsed field power for running small to mid sized agarose gels A PC running Pippin Pulse software is required The PC and gel tank must be provided by the user 1 1 Specifications Electrophoresis voltage 25 175 V Maximum current 200 mA Dimensions 7H X 20W X 24D cm 3H X 8W X 10D in Weight 1 1 kg 2 5 Ibs 1 2 Warranty The Pippin Pulse is subject to depot repair or replacement at the discretion of Sage Science The warranty period is 1 year from the date of shipment sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 2 0 About Pulsed Field Electrophoresis Pulsed field gel electrophoresis is a strategy for resolving large fragments of DNA for an2. User Manual Pippir j Pulse Electrophoresis Power Supply Software Version 1 32 Part No PPIO200 978 922 1832 www sagescience com support sagescience com Sage Science Inc Suite 3150 500 Cummings Center Beverly MA 01915 2014 Sage Science Inc All rights reserved Sage Science Pippin Prep BluePippin and Pippin Pulse are trademarks of Sage Science Inc SeaKem is a trademark of Lonza Group Ltd All other brands and names mentioned herein are property of their owners sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com Table of Contents JT OM ahd gore Ee d EE 3 LLSD Le 3 LZ WV AIIM Y ste inossrasG E E stole utes oacusacunawdccaeoneuey soon gunesoes E 3 2 0 About Pulsed Field Electrophoresis cccccccccccssssssecceessssseeeecessseeeeeeesssseeeceesesaeeeeeeesseaeeeeeens 4 3 0 Using the Pippin beet 6 GESEIS 6 BAR E 6 oo OG od f 0 0 0 e nen nee ee ee eee eee 7 6 1 Recommended gel conditions 7 6 1 1 KBB Gel pH 8 7 5 15kb and 10 48kb 00 0 a 7 612 TEGel PH S 0 S BOKO E 8 6 2 Pre set protocol example 1 5 15KD oo eeccccssecccessceceesceeeesececseceseesceseeeeeeeeecesseecessgaees 9 6 3 Pre set protocol example 2 10 A8kb eeccccssecccesececeseceeeneceeeeeceeeeeeeeeecessaecesseeeseees 10 6 4 Pre set protocol example 3 5 80kb 00 cecccccssecccesececesceeceneceeeeecessesceseeaeceseuecessueeessees 11
3. alues between 25 150 V A Forward Time at start of run 1 65 535 msec B Reverse Time at start of run 1 65 535 msec C Increment added to A at each step 0 255 msec D Increment added to B at each step 0 255 msec E Increment added to C at each step 0 255 msec F Increment added to D at each step 0 255 msec G Number of steps per cycle 1 65 535 9 2 Where to start The table below may be used on a guideline to begin developing protocols for gel analysis of large DNA These guidelines are based on 5 10 V cm gels and 0 5X electrophoresis buffer see Pre set Protocols Section 6 Running the Pippin Pulse The protocols that are highlighted in gray are pre set in the Pippin Pulse software the remaining protocols have not been tested but are provided as a starting recommendation Approx Pippin Pulse Parameters Resolved Size Range kb Run Time 8 hr 8 300 100 300 100 0 O 100 20 800 20 hr 9 300 100 0 O 60 20 51 40 1000 20 hr 10 300 100 300 100 0 O 250 40 2000 20 hr Fig 4 Pre set protocols highlighted in gray have been validated by Sage Science Other protocols are suggested starting protocols for running gels between 5 10 V cm 16 sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 9 3 Programming the Pippin Pulse 1 Turn the Pippin Pulse on by pressing the power button on the back panel 2 Launch th
4. alysis When running a typical direct current agarose gel fragments above 10 15kb can migrate in a non predictable manner that is not related to their size Pulsed field gels work by shuttling DNA back and forth in the gel effectively slowing down large DNA fragments that might otherwise run at the same rate as smaller ones By switching the direction of the electric field in a gel DNA will change its direction of migration Since smaller molecules can change direction faster than larger molecules more differentiated separation can be achieved by rapidly switching or pulsing the electric field A single pulse duration typically separates a relatively narrow range of DNA sizes For best results it is necessary to use a range of pulse times this is accomplished by using a ramp progressively increasing the forward and or reverse intervals from a lower limit to an upper limit The Pippin Pulse is programmed by entering a forward time step and reverse time step A ramp is programmed by adding a time increment to either or both steps An accelerated ramp can be achieved by adding additional time increment either or both increments Lastly the number of time steps is also entered and the steps will continuously cycle for the duration of the run Cycling allows the same protocol to be run for different lengths of time reproducibly Increment 600 500 400 msec Step 300 Step Time Forward 200 100 X x Step Time Reverse 0 Fi
5. e Pippin Pulse application by double clicking the Pippin Pulse Icon gd Pippin Pulse 00 00 00 i CE iS os FO ee a Ea 3 Press the EDIT Button The editing screen will appear over the Main Screen sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 4 Place the mouse cursor in the Editing Field Enter a name for the protocol under Waveform Type and add the values for each parameter New Protocol VT oe ee en SS Wavetorm Parameters 259 MSec 255 msec 25 5 mser Gi WM 5 Ifa previously written protocol is on the protocol list clicking on the protocol will make it appear in the editing field and it may be modified 10 48 kb Version A 6 Press ADD REPLACE to add the new protocol to the list If the same name appears in the Waveform Type field the protocol will be replaced with the newer one v Waveform Type V 0 5 15 kb 100 ao SU 7 Press ACCEPT to save the new list and return to the Main screen aff ACCEPT sage science Pippin Pulse User Manual Rev 18 978 922 1832 www sagescience com support sagescience com 10 0 Software Overview There are two screens in the Pippin Pulse software e The Main Screen lists the electrophoresis protocols provides run controls and displays the run Status e The Edit Screen allows the programming editing and deleting of electrophoresis pr
6. g 1 An example of time increments for the forward and reverse steps A 25 msec forward step with a 25 msec increment 10 msec reverse step with a 10 msec increment 20 Steps cycle sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com Increment Increment 2000 1500 msec Step 1000 Step Time Forward 500 x Step Time Reverse 0 Fig 2 An example of second increments added 5 msec was added to the forward increment and 3 msec increment was added to the reverse increment 4 cycles 2000 1500 msec Step 1000 e Step Time Forward 500 x Step Time Reverse 0 Fig 3 An Illustration of running four cycles of step of the previous protocol In this example each cycle is about 6 seconds in length Typical runs require 8 16 hours Note If the reverse time is not long enough large molecules may not completely change direction during the reversal and may actually migrate faster than shorter molecules This effect can be completely overcome by using a time ramp whose longest reverse time is long enough to separate all molecules of interest sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 3 0 Using the Pippin Pulse The Pippin Pulse software includes five preset protocols based on internal testing These are a good starting point to analyze fragments from 0 5 430kb Go to the Pre se
7. g file is saved after every run The file name is saved as PP_ year month day _ hour min sec txt The files are saved in a sub folder titled Pippin Pulse that is automatically placed in the computer s Documents folder during software installation The file lists the measured voltage and current values at 1 second intervals for the entire run gt A Public Documents rt Sage Science Pippin Pulse Logs 2012 12 v Search 2012 12 Organize v Include in library v Share with Burn New folder FJ e Ke E E Name Date modified Type Size BR Desktop e PP_2012 12 18_17 22 54_ 12 18 2012 5 23 PM Text Document A Downloads e PP_2012 12 19 15 52 16_ 12 19 2012 3 52PM Text Document File Edit Format View Help T s VUN ICMA 1 24 2 1 18 d 24 35 1 54 3 24 7 1 54 4 24 86 1 85 15 sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 9 0 Programming the Pippin Pulse 9 1 What is a Pippin Pulse protocol A Pippin Pulse protocol is a series of parameters that control the run voltage forward and reverse time steps and number of steps per cycle A more complete explanation of these parameters is described earlier in this manual About Pulsed Field Electrophoresis Section 3 The functions of the parameters are listed below and appear on the Editing screen of the Pippin Pulse software application V Voltage Electrophoresis voltage accept v
8. garose 10X TBE buffer Total 110 ml sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 6 2 Pre set protocol example 1 5 15kb Run time 8 hours Voltage 100 V Buffer 0 5X KBB see page 7 Agarose 0 75 Lonza SeakKem GOLD 110 ml 15k ais em die 10k 8k 6k 5k en 4k ER 3k E 2k ze ep 1k mm sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com 6 3 Pre set protocol example 2 10 48kb Run time 9 hours Voltage 80 V Buffer 0 5X KBB see page 7 Agarose 0 75 Lonza SeaKem GOLD 110 ml 10k 1kb cE 29 9 GE BR 170 oon 15 0 _ a 10 0 a e ome E Pa keng erm wem eso wm eme en om a ES e Ge sage science Pippin Pulse User Manual Rev support sagescience com 10 978 922 1832 www sagescience com 6 4 Pre set protocol example 3 5 80kb Run time 14 hours Voltage 80 V Buffer 0 5X TBE see page 8 Agarose 1 Lonza SeaKem GOLD 110 ml sage science Pippin Pulse User Manual Rev support sagescience com 11 978 922 1832 www sagescience com 6 5 Pre set protocol example 4 5 150kb Run time Voltage Buffer Agarose 16 hours 75V 0 5X KBB see page 8 0 75 Lonza SeaKem GOLD 110 ml 194 kb 145 5 kb 97 kb 48 5 kb sage science Pippin Pulse User Manual Rev support sage
9. om 6 0 Pre set protocols There are six preset fixed pulsed field protocols listed in the Pippin Pulse software These are highlighted in gray in the software and cannot be modified or deleted and indicate a fragment range over which DNA should be well resolved the DC Mode 25 direct current protocol is not hightlighted in gray and can be quickly edited and re saved These protocols were developed by Sage Science specifically to work with higher size range workflows with the BluePippin DNA size selection system Pippin Pulse v1 37 Waveform Table 5 80kb DC Mode DC Mode 25 6 1 Recommended gel conditions The pre set protocol gel images shown on the following pages were run using the following gel formulations in a 12 X 14 cm gel tank recommended Galileo 1214 Mini Gel Unit Sage Science PGB0001 Thermo Scientific Owl D2 Wide Gel System Fisher H 09528165 6 1 1 KBB Gel pH 8 7 Note 10X KBB buffer can be purchased from Sage Science Part No KBB1001 0 5X KBB running 10X buffer buffer g liter mM Tris base TAPS free acid EDTA free acid Lonza SeaKem GOLD 10X KBB buffer Total sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 6 1 2 TBE Gel pH 8 3 0 5X TBE running 10X buffer buffer Tris base Boric Acid 0 5M EDTA 0 5X TBE Buffer Total Lonza SeaKem GOLD dH20 950 ml 104 5 ml 1 A
10. otocols 10 1 The Main Screen When the Pippin Pulse application has been started the following screen will launch Electrophoresis Protocol List DO Pippin Pulse v 1 32 ei A v Ls GES Gs Gs Os Ce Ce res S Run Time Run Controls Run Status set by user 19 sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience com 10 2 The Edit Screen The Edit Screen is accessed by pressing the EDIT button and will appear over the Main Screen Electrophoresis Protocol List 0 5 15 kb 10 48 kb Wavetorm Parameters Editing field Description of Editing commands waveform parameters for programming sage science Pippin Pulse User Manual Rev 20 978 922 1832 www sagescience com support sagescience com 11 0 Pippin Pulse Blog A blog on Pippin Pulse can be found at the Sage Science website at http www sagescience com blog tips pippin pulse A link to this blog can also be found on the Pippin Pulse product page or by entering Pippin Pulse into the site s search bar We will endeavor to post the following e new protocols that we ve developed e references on gel electrophoresis and pulsed field e additional tools e user input and suggestions We invite you to check back periodically to see what s new sage science Pippin Pulse User Manual Rev 21
11. science com 48 5 kb 38 4 kb 33 5 kb 29 9 kb 24 5 kb 12 978 922 1832 www sagescience com 6 6 Pre set protocol example 5 5 430kb Run time 16 hours Voltage 75V Buffer 0 5X KBB see page 8 Agarose 0 75 Lonza SeaKem GOLD 110 ml 388 kb 291 kb 194 kb 48 5 kb sage science Pippin Pulse User Manual Rev support sagescience com 13 978 922 1832 www sagescience com support sagescience com 7 0 Running the Pippin Pulse 1 Set up the complete gel system load the DNA samples and connect the electrodes to the Pippin Pulse 2 Turn the Pippin Pulse on by pressing the power button on the front panel 3 Launch the Pippin Pulse application by double clicking the Pippin Pulse Icon gd Pippin Pulse i oS Os oe oa 4 Using your mouse pointer select the protocol or Waveform Type name to be run The selected protocol will be highlighted in blue 6 Press START sage science Pippin Pulse User Manual Rev 14 978 922 1832 www sagescience com support sagescience com 8 0 Monitoring a Pippin Pulse Run 1 When running the Pippin Pulse High Voltage Status will indicate a yellow color 2 The run voltage current values and run progress will be displayed High Voltage Status Measured Amplitude V Progress hh mm ss DL LI 8 1 Ending a Run To abort a run before the target duration press the ABORT button 8 2 Log Files A lo
12. t Protocols section of this manual Section 6 for the buffer formulation and conditions for these runs Go to Programming the Pippin Pulse Section 9 for recommendations and guidelines for developing new protocols 4 0 Unpacking The Pippin Pulse PPIO200 will arrive with the following components e Pippin Pulse electrophoresis power supply e Power supply e USB cable to connect PC to a Pippin Pulse e USB storage device contains the Pippin Pulse programming software e User manual this document 5 0 Set up e Remove the Pippin Pulse from its packaging e Connect the power supply to the unit and plug the power cable into an electrical outlet e Insert the USB cable into the USB port found in the back of the unit e Insert the other end of the USB cable into a USB port of a PC e Install the Pippin Pulse software on the PC Insert the USB storage device into an available port on a PC On the computer go to the USB device Computer Removable Disk E Open the Pippin Pulse folder Double click the setup executable file Follow the installer instructions CDM drivers and Pippin Pulse software After installation is complete re boot the computer O O O O 0 E The following icon will appear on the computer desktop Es Pippin Pulse Pippin Pulse Desktop Icon Double click the icon to launch the application sage science Pippin Pulse User Manual Rev 978 922 1832 www sagescience com support sagescience c
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