- 16. Bravo will then incubate the DNA with the beads for another 8 minutes \(total 10 min).
- 17. After incubation, Bravo will prompt you to move the ‘Beads + Binding’ plate from Pos4 to the magnet at Pos5.
- 17.1. Before clicking ‘Continue’, place the plate on the magnet at Pos5 and incubate for two minutes \(set a timer).
- 17.2. Click ‘Continue’ and the Bravo will incubate the plate on the magnet for 3.5 minutes set.
Note: This solution is very viscous and this additional time is necessary to allow all of the beads to settle on the magnet. This will help ensure we recover as much DNA as possible.
- 18. Next, Bravo will prompt you to remove the reservoir from the Pos9 and replace it with a 96 well deep-well reservoir \(Waste), and to remove the DNA plate from Pos6 and replace it with the
UV-treated Desulphonation buffer plate.
- 19. Next, Bravo will remove the binding and lysis buffer from DNA Plate \(Pos5):
- 19.1. Bravo will retrieve tips from the tip box at Pos3 and remove all of the liquid from the DNA plate \(‘Binding + Beads’ plate) at Pos5. Pause the protocol when Bravo hovers above Pos5 and uncover the plate. The tips will then be returned to Pos3.
Note: Be sure to check that all of the liquid has been aspirated as the ethanol percentage is critical in the subsequent steps.
- 20. Next, Bravo will perform two 80% ethanol washes and add desulphonation buffer:
- 20.1. Bravo will retrieve tips from the tip box at Pos2 and then aspirate 80% ethanol from Pos8. Pause the protocol when the Bravo machine hovers above Pos8 to remove the cover on the ethanol plate \(Pos8).
- 20.2. The ethanol will be gently dispensed into the DNA plate \(Pos5).
- 20.3. Bravo will let the ethanol solution and the beads incubate for 30 seconds before aspirating all of the liquid from the wells \(Pos5) and dispensing the waste fluid at Pos9.
- 20.4. Bravo will then perform a second identical ethanol wash.
- 20.5. After the waste from the 2nd ethanol wash is dispensed at Pos9, pause the protocol when the Bravo hovers above the desulphonation plate \(Pos6). Remove the desulphonation plate cover \(Pos6) and cover the ethanol plate \(Pos8).
- 20.6. Bravo will retrieve 60uL of desulphonation buffer from Pos6 and dispense it into the DNA plate at Pos5. The tips will then be returned to Pos2.
- 21. Bravo will incubate the DNA with the desulphonation buffer for 15 minutes.
Cover the DNA plate \(Pos5).
- 22. After the 15 minutes incubation, Bravo will prompt with a pop-up message. Follow the instructions and remove the plate cover on Pos5 before clicking ‘Continue’.
- 23. Bravo will discard the waste fluid and perform two ethanol washes:
- 23.1. Bravo will retrieve tips from the tip box at Pos2 and then aspirate the used desulphonation buffer from Pos5 and discard the waste fluid at Pos9.
- 23.2. Bravo will then aspirate 80% ethanol from Pos8 to perform the first wash. Pause the protocol when the Bravo machine hovers above Pos8 to remove the cover on the ethanol plate \(Pos8).
- 23.3. The ethanol will be gently dispensed onto the DNA plate \(Pos5).
- 23.4. Bravo will let the ethanol solution and the beads incubate for 30 seconds before aspirating all of the liquid from the wells \(Pos5) and dispensing the waste fluid at Pos9.
- 23.5. Bravo will then perform a second identical ethanol wash.
- 23.6. After the waste from the 2nd ethanol wash is discarded, let the DNA plate dry for one minute uncovered \(start a timer for one minute). The tips will then be returned to Pos2.
- 23.7. Cover the DNA plate \(Pos5) when the timer alarm goes off and let the DNA dry \(covered) for two additional minutes.
- 24. While the beads are drying, replace the plate at Pos6 with your UV-treated QEB plate.
- 25. After two minutes, Bravo will prompt you with a pop-up message. Remove the cover on the QEB plate \(Pos6) before clicking ‘Continue’.
- 26. Bravo will then add 15uL of QEB \(Pos6) to your DNA plate \(Pos4):
- 26.1. Bravo will retrieve tips from the tip box at Pos1 and aspirate 15uL of QEB from Pos6.
- 26.2. Pause the protocol when the Bravo hovers above Pos4 to dispense the QEB. Remove the cover from the DNA plate and move the plate from Pos5àPos4.
- 26.3. Bravo will dispense the QEB into the DNA plate at Pos4 and mix to resuspend the beads.
- 27. After the beads are re-suspended in EB buffer, cover the plate with a BioRad PCR cover and place the plate in the Eppendorf thermomixer at 56˚C for 15 minutes at 2000 rpm \(according to pop-up message). Tape the plate down to the incubator to prevent the plate from flying off.
- 27.1. Set a timer for five minutes and when the alarm goes off, briefly turn the thermomixer down to 1700 rpm to allow the liquid near to the top of the vortex to reincorporate into the larger volume. Then turn the thermomixer back up to 2000 rpm.
- 27.2. Set another timer for five minutes and repeat.
- 28. While the plate is incubating start preparation for 1st DNA synthesis.
- sc 1st DNA Synthesis
- 1. Retrieve reagents needed for 1st DNA synthesis \(except enzymes) from -20° C freezer, thaw them and place them on ice.
- 2. Working on ice, prepare the DNA denaturation mix according to the following calculator:
- 3. Mix the pre-denaturation mix very well by repeated pulse-vortexing, spin down in a minifuge, and keep it on ice.
- 4. Aliquot 72uL of the mix into one 12-well strip tube \(7.25 uL**8rows+14uLdead volume=72uL).
- 5. Change the tip box at position 2 on Bravo.
- 6. Pause the program and update the tip editor with Box-position2.
- 7. Place the master mix strip-tube on row A of a chilled cool-rack at position 7 on Bravo.
- 8. Aliquot 7.25uL of pre-denaturation mix into each will of a new LoBind plate. Place this plate on a chilled cool-rack at position 6 on Bravo.
- 9. After the 56°C incubation is complete, place the “Binding + Beads” plate in a thermocycler and close the hot lid. Keep it in for 5 seconds to remove any condensation on the tape pad. If there is any droplet on the side of the wells, spin the plate down shortly for 3~4 sec.
- 10. Place the “Binding + Beads” plate back on the Bravo deck: on top of the magnet in position 5.
- 11. The robot will first aliquot the master mix brew from the 12-well strip tube into the new LoBind plate 7.25 uL/well at position 6. Then it will transfer the elute DNA 15 uL/well into the brew plate and mix.
- 12. After the program completes, seal the plate with BioRad PCR cover and spin down the “scPBAL PR” plate for 1 minute at 4˚C at 3000 rpm.
- 13. Incubate the “scPBAL PR” plate at 98°C for 1 minute in a thermocycler and immediately spin down the plate at 3000 rpm for 1 minutes at 4˚C. Place this plate on ice, keep it COLD.
- 14. Working on ice, prepare post-denaturation mix according to the following calculator:
- 15. Vortex the buffer before addition to the brew.
- 16. Remove the enzyme from -20°C freezer and place it in the chilled tube coolrack for transport to the PCR workstation.
- 17. Mix the enzyme by flicking the tube, spin down in a minifuge, and place it back in the chilled tube coolrack.
- 18. When you pipette the enzyme, aspirate slowly. After dispensing the enzyme into the brew, wash the tip by pipetting up and down. Place the brew back on ice. Return the enzyme back into the -20°C freezer before proceeding.
- 19. Mix the brew very well by multiple pulse-vortexing and keep it on ice.
- 20. Aliquot 27uL per well of the post-denaturation mix into a row of the “RV” plate. Cover and if necessary spin the plate down at 3000rpm for 1min, 4°C to collect the liquid on the bottom. Place this plate in a -20˚C chilled coolracks.
- 21. Place the “scPBAL RP” plate on the other -20˚C chilled plate coolrack.
- 22. Using P20 multichannel, add 3uL per well of the post-denaturation mix into each row of the “scPBAL RP” plate. After each row addition, set P20 multichannel to 10uL, mix each row up and down 10 times. Change tips between rows.
- 23. After mixing of the last row, seal the plate with BioRad PCR cover, spin the plate down at 3000 rpm, 4°C for 1min.
- 24. Place the plate in the thermocycler, close the lid and run the program pbat-rand-a:
4°C for 10 minutes
4°C to 37°C ramping at 4°C/minute
37°C for 30 minutes
98°C for 1 minutes
4˚C forever
- 25. Place the coolracks back in the -20°C freezer to chill for the next step.
- sc 2nd DNA synthesis
- 1. About 15 minutes before the program is finished start preparing for the second round of random priming.
- 2. Working on ice, prepare 2nd DNA synthesis mix according to the following calculator:
- 3. All reagents \(except enzymes) must be pulse-vortexed and spin down in a minifuge before addition to the brew. Enzymes should be added last.
- 4. When ready to add the enzyme, pulse-vortex the brew multiple times, then spin down in a minifuge, and place back on ice.
- 5. Remove the Klenow exo – \(50U/uL) from -20°C freezer and place it in the chilled tube cool rack for transport to the PCR workstation.
- 6. Mix each enzyme by flicking the tube, then spin down in a minifuge and place it back in the chilled tube cool rack.
- 7. When pipetting the enzyme, aspirate slowly to assure an accurate volume transfer. Enzyme storage buffers contain glycerol, which makes the solution more viscous than water.
- 8. After dispensing the enzyme into the brew, wash the tip by pipetting up and down. After enzyme addition, place the brew immediately back on ice. Return the enzyme back into the -20°C freezer before proceeding.
- 9. Mix the entire 2nd DNA synthesis mix very well by multiple pulse-vortexing, spin down in a minifuge and place it back on ice.
- 10. Once the pbat-rand program is finished, immediately spin down the “scPBAL RP” plate for 2 minutes at 4˚C at 3000 rpm. Place the plate on ice.
- 11. Aliquot 45uL/well of the prepared mix into row F of “RV” plate. Cover and keep it on ice. If needed, spin down for 1min at 3000 rpm at 4˚C. Place this plate in a -20˚C chilled coolrack.
- 12. Place the “scPBAL RP” plate on another -20˚C chilled coolrack.
- 13. Using a P20 multichannel, add 5 uL per well of the 2nd DNA synthesis mix to each row of the “scPBAL RP” plate. After each addition, set the multichannel to 15 uL and mix up and down at least 10X, lifting your pipet when dispensing and aspirating from the bottom of the well. Change tips between rows.
- 14. Seal the plate with a BioRad PCR cover and spin down for 1 minute at 4˚C at 3000 rpm.
- 15. Place the plate in the thermocycler, close the lid, and run the program pbat-rand-b:
4°C for 30 seconds
4°C to 37°C ramping at 1°C/15 seconds
37°C for 30 minutes
70°C for 10 minutes
4˚C forever
- 16. After the program finishes, spin down the plate at 3000 rpm for 1min at 4˚C.
- 17. In hood, change the PCR cover to Aluminum Cover. Seal and label with name and date, and store at -20˚C overnight.
Clean up your workstation. Remove garbage. This concludes Day 1 of this protocol.
DAY 2
- SeraMag Bead Cleanup \(BCU) and End Repair/Phosphorylation
- 1. Pre-chill two coolracks at -20˚C. Put on a new disposable lab coat, clean workstation and Bravo.
- 2. Take out 20% PEG SeraMag beads from 4°C \(6 ml is required for one full 96-well plate). Mix by vortexing and leave them to warm at room temperature for at least 30 minutes.
- 3. Ensure that the correct 96 LT head is on the Bravo.
- 4. Turn on the Bravo computer and open VWorks. Wait for the program to finish loading. Press the login button at the top and enter your account information.
- 5. Lift the hood sash until it is just above the red arrows. Turn on the light and the fan. Turn on the switch on the right side of the Agilent Bravo and wait for a steady blue indicator light.
- 6. Clean the Bravo deck with RNase or DNA Away.
- 7. Prepare 50mL of 70% ethanol \(mix 35mL of 100% ethanol with 15mL of ultrapure water).
- 8. Pour Qiagen EB buffer into a clean reservoir. Aliquot 100 uL of EB buffer into each well of a new AB1400 plate. Label this plate the “EB” plate.
- 9. Retrieve “scPBAL RP” plate from -20˚C. Spin down at 3000 rpm for 1min, 4°C.
- 10. Measure the volume of three random DNA wells \(should be 30 uL, but may be less due to evaporation) and using P20 multichannel add sufficient EB \(usually 21-24 uL) to reach 50 uL/well. After each row addition, mix well by pipetting up and down 10X. Change tips between rows.
Note: It is better to have >50 uL than to have <50 uL in this step.
- 11. Make sure that the DNA/reaction plate is spun down at 3000rpm for 1min, 4°C before placing it on the Bravo deck.
- 12. Once SeraMag beads reach room temperature, aliquot them into one row of a 450uL Axygen plate.
- 13. Using P200 multichannel, aliquot 55 uL of beads into each well of a new AB1400 plate. Label this plate the “beads”. If there are beads on the side of the well, short spin of 4-5 seconds is allowed. Do not spin down beyond that.
- 14. Open the PBAL form. File > Open and select: C:\VWorks Workspace\NGS\Forms\PBAL.VWForm. To be able to see the form you must change the file type at the bottom right of the window.
- 15. In the form, from the ‘select protocol’ drop down list choose “Ampure XP Bead Cleanup-tony”. Hit the ‘Display Initial Setup’ button on the form.
- 16. Set up the deck on the Bravo deck according to the setup diagram on the screen which should match the image below:
Positions 1-3: New 96-well tip boxes \(full plate of tips)
Position 4: Coolrack + “DNA” plate \(make sure the plate us spun down)
Position 5: low volume magnet \(Magnum FLX)
Position 6: Coolrack + “beads” plate
Position 7: empty
Position 8: 96 well 450V reservoir
Position 9: Axygen LP reservoir for 70% ethanol
- 17. Enter the number of rows to be processed \(8 for a full plate) and set the variables as follows:
DNA volume = 50 uL
Bead volume = 50 uL
Elution volume = 35 uL
Ethanol volume = 150 uL
- 18. Check the Bravo deck if all of the items are present and placed correctly. Make sure that all of the covers have been removed from plates and tip boxes before proceeding.
- 19. At the bottom of the NGS form, hit the Start button. The Bravo will initialize. If an error about a plate being detected in the gripper pops up, hit ‘Ignore’. Another error will pop up shortly thereafter. Hit ‘Retry’. The Bravo should now begin the protocol.
- 20. Before any tasks begin, VWorks will ask you to configure the tip state editor. Pause the protocol, and go to Tools > Tip State Editor.
- 21. Click the ‘…’ button on the right of the tip box name and change the tip state to match the tip box on the deck. Repeat for all tip boxes then hit ‘OK’ and ‘Continue’.
Note: While the protocol is being executed watch the Bravo to make sure that all volumes are being correctly pipetted. For example, there should be no empty space in the tips when beads are being added to the DNA.
- 22. The program will add the beads \(Pos6) to the DNA plate \(Pos4). Bravo will retrieve tips from Pos3 and transfer 50uL of beads from Pos6 to the DNA plate at Pos4 and mix. The tips will then be returned to Pos3.
- 23. After bead addition, during the incubation, pause the protocol and cover the DNA plate with a plastic cover.
- 24. The program will aliquot the ethanol into the 96 well reservoir \(Pos9àPos8)
- 24.1. Bravo will retrieve tips from the tip box at Pos2 and aliquot ethanol from the reservoir \(Pos9) into the ethanol plate \(Pos8). Pause the program when the machine hovers above Pos9 to pour the fresh 70% Ethanol into the reservoir. The tips will be returned to Pos2 after the last of the ethanol is aliquoted.
- 24.2. After ethanol aliquoting, pause the program and cover the ethanol plate at Pos8.
- 25. The program will then incubate the DNA plate with the beads for 8 minutes \(10 minutes total).
- 26. After incubation, the program will prompt you to put the plate on the magnet and replace the ethanol reservoir from Pos9 with an empty Axygen 450 waste plate.
- 26.1. Place the plate on the magnet at Pos5 and incubate for 5 minutes.
Note: Check the walls of the wells to ensure that all of the liquid is at the bottom of the well before placing the plate on the magnet. Spin for 3-4s in the centrifuge if this condition is not met.
- 27. Next, Bravo will remove the waste fluid from the DNA plate \(Pos5).
- 27.1. Bravo will retrieve tips from the tip box at Pos3 and proceed to remove all of the liquid from the DNA plate at Pos5. Pause the protocol when Bravo hovers above Pos5 and uncover the plate.
- 27.2. Bravo will dispense the waste fluid at Pos9. The tips will then be returned to Pos3.
- 28. Next, Bravo will perform two 70% ethanol washes.
- 28.1. Bravo will retrieve tips from the tip box at Pos2 and then aspirate 70% ethanol from Pos8. Pause the protocol when the Bravo machine hovers above Pos8 to remove the cover on the ethanol plate \(Pos8).
- 28.2. The ethanol will be gently dispensed into the DNA plate \(Pos5).
- 28.3. Bravo will let the ethanol solution and the beads incubate for 30 seconds before aspirating all of the liquid from the wells \(Pos5) and dispensing the waste fluid at Pos9.
- 28.4. Bravo will then perform a second identical ethanol wash.
- 28.5. After the waste from the 2nd ethanol wash is dispensed at Pos9, set a timer for one minute. The tips will then be returned to Pos2.
- 29. When the timer alarms, cover the plate \(Pos5) and let the plate dry for two additional minutes.
Note: Before continuing, inspect the reaction plate to make sure that all of the traces of ethanol are evaporated. The beads should be completely dry but not cracked. Make sure the beads are not over-dried as that reduces DNA recovery.
- 30. Next, Bravo will prompt you to move the DNA plate from Pos5 to Pos4 and to place the QEB plate at Pos6. Do not move the DNA plate yet, but place the QEB plate at Pos6. Remove the cover on the QEB plate and click ‘Continue’.
- 31. The program will then add 35 uL QEB \(Pos6) to the DNA plate \(Pos4) and incubate for a minimum of four minutes.
- 31.1. Bravo will retrieve tips from the tip box at Pos1 and aspirate QEB from Pos6.
- 31.2. Bravo will then dispense the QEB into the DNA plate at Pos4. Pause the protocol when the machine hovers above Pos4. Uncover the DNA plate \(Pos5) and move it to Pos4.
- 31.3. The tips will then be returned to Pos1.
- 31.4. Cover the DNA plate \(Pos4) when the machine is no longer in motion.
- 32. After the EB buffer has been added and mixed with dried beads pause the program and in a 2.0mL tube prepare the End-repair master mix according to the following calculator:
- 33. All reagents \(except enzymes) must be pulse-vortexed and spin down in a minifuge before addition to the brew. Enzymes should be added last. When ready to add the enzymes, pulse-vortex the end repair master mix multiple times, then spin down in a minifuge, and place back on ice.
- 34. Remove the enzymes from -20°C freezer and place them in the chilled tube cool rack for transport to the PCR workstation. Mix each enzyme by flicking the tube, then spin down in a minifuge and place it back in the chilled tube cool rack.
- 35. When pipetting the enzyme, aspirate slowly to assure an accurate volume transfer. Enzyme storage buffers contain glycerol, which makes the solution more viscous than water. After dispensing the enzyme into the brew, wash the tip by pipetting up and down.
Note: Never double dip into the solution with the same tip.
- 36. After enzyme addition place the brew immediately back on ice. Return the enzymes back into the -20°C freezer before proceeding.
- 37. Gently pulse-vortex the brew multiple times to assure an even distribution of all of the components. Pulse-vortexing means that you stop vortexing every time a full vortex is formed in the tube. Spin down the brew in a minifuge and immediately place it back on ice.
- 38. Working on ice, aliquot 143 uL of the mix into one 12-well strip tube.
- 39. Change the tip box at position 2 on Bravo and pause the program and update the tip editor with Box-position2.
- 40. Place the master mix strip-tube on row A of a chilled cool-rack at position 7 on Bravo. Place the elution plate back on the Bravo deck: on top of the magnet in Pos5.
- 41. The robot will first aliquot the master mix brew from the 12-well strip tube into the new LoBind plate 15 uL/well at position 6. Bravo will transfer \(Pos5) the DNA elution into the brew plate \(Pos6) and mix.
- 42. After the program completes, cover the plate and spin down the plate for 1 minute at 4˚C at 3000 rpm.
- 43. Incubate at room temperature for 30 minutes then proceed to the next step.
- Bead Cleanup and A-Tailing
- 1. Set up the Bravo exactly as stated in Bead Clean-up step 10 except with the following change in the parameters:
DNA volume = 50 uL
Bead volume = 50 uL
Elution volume = 25 uL
Ethanol volume = 150 uL
- 2. Make sure that the DNA/reaction plate is spun down at 3000rpm for 1min, 4C before placing it on the Bravo deck.
- 3. Aliquot SeraMag beads into one row of a 450uL Axygen plate.
- 4. Using P200 multichannel, aliquot 55 uL of beads into each row of a “beads” plate. If there are beads on the side of the well, short spin of 4-5 seconds is allowed.
- 5. Check the Bravo deck if all of the items are present and placed correctly. Make sure that all of the covers have been removed from plates and tip boxes before proceeding.
- 6. Start the protocol. Before any tasks begin, VWorks will ask you to configure the tip state editor. Pause the protocol, and go to Tools > Tip State Editor.
- 7. Click the ‘…’ button on the right of the tip box name and change the tip state to match the tip box on the deck. Repeat for all tip boxes then hit ‘OK’ and ‘Continue’.
Note: While the protocol is being executed watch the Bravo to make sure that all volumes are being correctly pipetted. For example, there should be no empty space in the tips when beads are being added to the DNA.
- 8. The program will add the beads \(Pos6) to the DNA plate \(Pos4).
- 8.1. Bravo will retrieve tips from Pos3 and transfer 50uL of beads from Pos6 to the DNA plate at Pos4 and mix. The tips will then be returned to Pos3.
- 8.2. Pause the protocol and cover the DNA plate.
- 9. The program will aliquot the ethanol into the 96 well reservoir \(Pos9àPos8)
- 9.1. Bravo will retrieve tips from the tip box at Pos2 and aliquot ethanol from the reservoir \(Pos9) into the ethanol plate \(Pos8). Pause the program when the machine hovers above Pos9 to pour the fresh 70% Ethanol into the reservoir. The tips will be returned to Pos2 after the last of the ethanol is aliquoted.
- 9.2. Pause the program and cover the ethanol plate at Pos8.
- 10. The program will then incubate the DNA plate with the beads for 8 minutes \(10 minutes total).
- 11. After incubation, the program will prompt you to put the plate on the magnet and replace the ethanol reservoir from Pos9 with an empty Axygen 450 waste plate.
- 11.1. Place the plate on the magnet at Pos5 and incubate for two minutes \(set a timer).
- 11.2. Click ‘Continue’ when the timer alarms and incubate for an additional 3.5 minutes.
Note: Check the walls of the wells to ensure that all of the liquid is at the bottom of the well before placing the plate on the magnet. Spin for 3-4s in the centrifuge if this condition is not met.
- 12. Next, Bravo will remove the waste fluid from the DNA plate \(Pos5).
- 12.1. Bravo will retrieve tips from the tip box at Pos3 and proceed to remove all of the liquid from the DNA plate at Pos5. Pause the protocol when Bravo hovers above Pos5 and uncover the plate.
- 12.2. Bravo will dispense the waste fluid at Pos9. The tips will then be returned to Pos3.
- 13. Next, Bravo will perform two 70% ethanol washes.
- 13.1. Bravo will retrieve tips from the tip box at Pos2 and then aspirate 70% ethanol from Pos8. Pause the protocol when the Bravo machine hovers above Pos8 to remove the cover on the ethanol plate \(Pos8).
- 13.2. The ethanol will be gently dispensed into the DNA plate \(Pos5).
- 13.3. Bravo will let the ethanol solution and the beads incubate for 30 seconds before aspirating all of the liquid from the wells \(Pos5) and dispensing the waste fluid at Pos9.
- 13.4. Bravo will then perform a second identical ethanol wash.
- 13.5. After the waste from the 2nd ethanol wash is dispensed at Pos9, set a timer for one minute. The tips will then be returned to Pos2.
- 14. When the timer alarms, cover the plate \(Pos5) and let the plate dry for two additional minutes.
Note: Before continuing, inspect the reaction plate to make sure that all of the traces of ethanol are evaporated. The beads should be completely dry but not cracked. Make sure the beads are not over-dried as that reduces DNA recovery.
- 15. Next, Bravo will prompt you to move the DNA plate from Pos5 to Pos4 and to place the QEB plate at Pos6. Do not move the DNA plate yet, but place the QEB plate at Pos6. Remove the cover on the QEB plate and click ‘Continue’.
- 16. The program will then add 25 uL QEB \(Pos6) to the DNA plate \(Pos4) and incubate for a minimum of four minutes.
- 16.1. Bravo will retrieve tips from the tip box at Pos1 and aspirate QEB from Pos6.
- 16.2. Bravo will then dispense the QEB into the DNA plate at Pos4. Pause the protocol when the machine hovers above Pos4. Uncover the DNA plate \(Pos5) and move it to Pos4.
- 16.3. The tips will then be returned to Pos1.
- 16.4. Cover the DNA plate \(Pos4) when the machine is no longer in motion.
- 17. After the EB buffer has been added and mixed with dried beads, in a 1.5mL tube prepare A-addition brew according to the following calculator:
- 18. Aliquot 95 uL of the mix into one 12-well strip tube.
- 19. Change the tip box at position 2 on Bravo. Pause the program and update the tip editor with Box-position2.
- 20. Place the master mix strip-tube on row A of a chilled cool-rack at position 7 on Bravo. Place a LoBind plate on a chilled cool-rack at position 6 on Bravo.
- 21. Place the elution plate back on the Bravo deck: on top of the magnet in position 5.
- 22. The robot will first aliquot the master mix brew from the 12-well strip tube into the new LoBind plate 10 uL/well at position 6. Then it will transfer \(Pos5) the elute DNA 25 uL/well into the brew plate \(Pos6) and mix.
- 23. After the program completes, seal the plate with a BioRad PCR cover and spin down the “A-addition” plate at 4˚C at 3000 rpm for 1 minute.
- 24. Incubate the final reaction \(35 uL total volume) at 37˚C for 30 minutes and then inactivate at 70˚C for 10 minutes.
Note: Do not cover with the Thermocycler lid. Loosely cover with Thermomixer lid.
- 25. While waiting, prepare for the next step.
- sc PBAL direct ligation
- 1. Retrieve the 5X Quick Ligation Buffer from -20˚C and thaw at room temperature then transfer to ice. Mix very well before use. This reagent contains high concentration of PEG, which makes it more difficult to pipette. Take an extra care to pipette this reagent slowly to assure accuracy of volume transfer.
- 2. Retrieve one plate single use aliquot of 10 uM PE Adapter. Prepare 300uL of 1 uM Adapter \(1/10 dilution, 30 uL stock + 270uL EB) by diluting with EB buffer. Aliquot the diluted 1 uM adapter into one row of a 96-well plate, 24uL/well. Cover the plate and keep it on ice.
- 3. Prepare the ligation brew in one 5 mL tube according to the following calculator:
- 4. All reagents \(except enzymes) must be pulse-vortexed and spin down in a minifuge before addition to the brew. Enzymes should be added last. When ready to add the enzymes, pulse-vortex the end repair master mix multiple times, then spin down in a minifuge, and place back on ice.
- 5. Remove the enzymes from -20°C freezer and place them in the chilled tube cool rack for transport to the PCR workstation. Mix each enzyme by flicking the tube, then spin down in a minifuge and place it back in the chilled tube cool rack.
- 6. When pipetting the enzyme, aspirate slowly to assure an accurate volume transfer. Enzyme storage buffers contain glycerol, which makes the solution more viscous than water.
- 7. After dispensing the enzyme into the brew, wash the tip by pipetting up and down. Do not double dip with the same tip into a stock solution. After enzyme addition, place the brew immediately back on ice.
- 8. Return the enzymes back into the -20°C freezer before proceeding.
- 9. Gently pulse vortex the brew multiple times to assure an even distribution of all of the components. Pulse vortexing means that you stop vortexing every time a full vortex is formed in the tube. Briefly spin down the brew in a minifuge and place it back on ice.
- 10. Aliquot 200 uL of the mix into one 12-well strip tube.
- 11. Change the tip box at position 2 on Bravo. Pause the program and update the tip editor with Box-position2.
- 12. Place the master mix strip-tube on row A of a chilled cool-rack at position 7 on Bravo.
- 13. Place the elution plate back on the Bravo deck: on top of the magnet in position 5.
- 14. Manually add 2 uL adaptor from the reservoir row to each well of new LoBind plate using P20 multichannel pipette. Label the plate “PBAL Ligation”. Place the plate on a chilled cool-rack at position 6 on Bravo.
- 15. The robot will first aliquot the master mix brew from the 12-well strip tube into the new LoBind plate 23 uL/well at position 6. Click ‘Continue’ and Bravo will transfer \(Pos5) the DNA elution 35 uL/well into the brew plate \(Pos6) and mix.
- 16. Once the program finishes, cover the ‘PBAL Ligation’ plate at Pos6 and move it to the laminar flow hood. Add 23 uL of the ligation master mix into each well via P200 multichannel pipette.
- 17. Use the P200 multichannel pipette to mix. Set the pipette to 40 uL and then aspirate the reaction mixture from the bottom of each well and slightly lift the tip of the pipette before dispensing on the top. Repeat this ten times to ensure thorough mixing.
Note: Pipet slowly to avoid bubbles.
- 18. Cover the plate with an aluminum cover, and spin down at 3000rpm for 1min at 4C.
- 19. Incubate the “PBAL Ligation” plate at room temperature overnight. Label the date and sample name clearly.
- 20. Clean up your single cell PCR work-station and Bravo. Turn off all of the used equipment.
- 21. Enter all of the data in your laboratory notebook. This concludes Day 2 of this protocol.
Clean up your workstation. Remove garbage. This concludes Day 2 of this protocol.
DAY 3
- 1st Bead Cleanup After Ligation
- 1. Take out 20% PEG SeraMag beads from 4°C \(enough for one full 96-well beads clean-up will be 6 tubes), mix by vortexing and leave them to warm at room temperature for at least 30 minutes. Before proceeding make sure that the SeraMag bead solution fully reached room temperature. Using a cold solution results in reduced DNA recovery.
- 2. Aliquot 55 uL of beads into each well of a new plate. Label this plate the “beads” plate. Do NOT use a reservoir for this step as the beads will settle and the PEG concentration will not be consistent.
- 3. Aliquot 120 uL of EB buffer into each well of a new plate. Label this plate the “EB” plate.
- 4. Set up the bravo exactly as stated in Bead-Cleanup and End-Repair \(step 10) except with the following parameters:
DNA volume = 60 uL
Bead volume = 48 uL
Elution volume = 50 uL
- 5. The program will add the beads \(Pos6) to the DNA plate \(Pos4).
- 5.1. Bravo will retrieve tips from Pos3 and transfer 50uL of beads from Pos6 to the DNA plate at Pos4 and mix. The tips will then be returned to Pos3.
- 5.2. Pause the protocol and cover the DNA plate.
- 6. The program will aliquot the ethanol into the 96 well reservoir \(Pos9àPos8)
- 6.1. Bravo will retrieve tips from the tip box at Pos2 and aliquot ethanol from the reservoir \(Pos9) into the ethanol plate \(Pos8). Pause the program when the machine hovers above Pos9 to pour the fresh 70% Ethanol into the reservoir. The tips will be returned to Pos2 after the last of the ethanol is aliquoted.
- 6.2. Pause the program and cover the ethanol plate at Pos8.
- 7. The program will then incubate the DNA plate with the beads for 8 minutes \(10 minutes total).
- 8. After incubation, the program will prompt you to put the plate on the magnet and replace the ethanol reservoir from Pos9 with an empty Axygen 450 waste plate.
- 8.1. Place the plate on the magnet at Pos5 and incubate for two minutes \(set a timer).
- 8.2. Click ‘Continue’ when the timer alarms and incubate for an additional 3.5 minutes.
Note: Check the walls of the wells to ensure that all of the liquid is at the bottom of the well before placing the plate on the magnet. Spin for 3-4s in the centrifuge if this condition is not met.
- 9. Next, Bravo will remove the waste fluid from the DNA plate \(Pos5).
- 9.1. Bravo will retrieve tips from the tip box at Pos3 and proceed to remove all of the liquid from the DNA plate at Pos5. Pause the protocol when Bravo hovers above Pos5 and uncover the plate.
- 9.2. Bravo will dispense the waste fluid at Pos9. The tips will then be returned to Pos3.
- 10. Next, Bravo will perform two 70% ethanol washes.
- 10.1. Bravo will retrieve tips from the tip box at Pos2 and then aspirate 70% ethanol from Pos8. Pause the protocol when the Bravo machine hovers above Pos8 to remove the cover on the ethanol plate \(Pos8).
- 10.2. The ethanol will be gently dispensed into the DNA plate \(Pos5).
- 10.3. Bravo will let the ethanol solution and the beads incubate for 30 seconds before aspirating all of the liquid from the wells \(Pos5) and dispensing the waste fluid at Pos9.
- 10.4. Bravo will then perform a second identical ethanol wash.
- 10.5. After the waste from the 2nd ethanol wash is dispensed at Pos9, set a timer for one minute. The tips will then be returned to Pos2.
- 11. When the timer alarms, cover the plate \(Pos5) and let the plate dry for two additional minutes.
Note: Before continuing, inspect the reaction plate to make sure that all of the traces of ethanol are evaporated. The beads should be completely dry but not cracked. Make sure the beads are not over-dried as that reduces DNA recovery.
- 12. Next, Bravo will prompt you to move the DNA plate from Pos5 to Pos4 and to place the QEB plate at Pos6. Do not move the DNA plate yet, but place the QEB plate at Pos6. Remove the cover on the QEB plate and click ‘Continue’.
- 13. The program will then add 50 uL QEB \(Pos6) to the DNA plate \(Pos4) and incubate for a minimum of four minutes.
- 13.1. Bravo will retrieve tips from the tip box at Pos1 and aspirate QEB from Pos6.
- 13.2. Bravo will then dispense the QEB into the DNA plate at Pos4. Pause the protocol when the machine hovers above Pos4. Uncover the DNA plate \(Pos5) and move it to Pos4.
- 13.3. The tips will then be returned to Pos1.
- 13.4. Cover the DNA plate \(Pos4) when the machine is no longer in motion.
- 14. Place a new LoBind plate at Pos6. Bravo should elute the supernatant directly into new plate.
- 15. Take the final eluted product and use it as the input DNA for the next step.
- 2nd Bead cleanup after ligation and PCR amplification of library
- 1. Aliquot 55 uL of beads into each well of the “beads” plate. Do NOT use a reservoir for this step as the beads will settle and the PEG concentration will not be consistent.
- 2. Set up the Bravo exactly as stated in Bead-Cleanup and End-Repair \(step 10) except with the following parameters:
DNA volume = 50 uL
Bead volume = 50 uL
Elution volume = 25 uL
- 3. Start the protocol. Replace the “beads” plate with the “EB” plate after the beads have been added to the DNA plate.
- 4. The program will add the beads \(Pos6) to the DNA plate \(Pos4).
- 4.1. Bravo will retrieve tips from Pos3 and transfer 50uL of beads from Pos6 to the DNA plate at Pos4 and mix. The tips will then be returned to Pos3.
- 4.2. Pause the protocol and cover the DNA plate.
- 5. The program will aliquot the ethanol into the 96 well reservoir \(Pos9àPos8)
- 5.1. Bravo will retrieve tips from the tip box at Pos2 and aliquot ethanol from the reservoir \(Pos9) into the ethanol plate \(Pos8). Pause the program when the machine hovers above Pos9 to pour the fresh 70% Ethanol into the reservoir. The tips will be returned to Pos2 after the last of the ethanol is aliquoted.
- 5.2. Pause the program and cover the ethanol plate at Pos8.
- 6. The program will then incubate the DNA plate with the beads for 8 minutes \(10 minutes total).\
- 7. After incubation, the program will prompt you to put the plate on the magnet and replace the ethanol reservoir from Pos9 with an empty Axygen 450 waste plate.
- 7.1. Place the plate on the magnet at Pos5 and incubate for two minutes \(set a timer).
- 7.2. Click ‘Continue’ when the timer alarms and incubate for an additional 3.5 minutes.
Note: Check the walls of the wells to ensure that all of the liquid is at the bottom of the well before placing the plate on the magnet. Spin for 3-4s in the centrifuge if this condition is not met.
- 8. Next, Bravo will remove the waste fluid from the DNA plate \(Pos5).
- 8.1. Bravo will retrieve tips from the tip box at Pos3 and proceed to remove all of the liquid from the DNA plate at Pos5. Pause the protocol when Bravo hovers above Pos5 and uncover the plate.
- 8.2. Bravo will dispense the waste fluid at Pos9. The tips will then be returned to Pos3.
- 9. Next, Bravo will perform two 70% ethanol washes.
- 9.1. Bravo will retrieve tips from the tip box at Pos2 and then aspirate 70% ethanol from Pos8. Pause the protocol when the Bravo machine hovers above Pos8 to remove the cover on the ethanol plate \(Pos8).
- 9.2. The ethanol will be gently dispensed into the DNA plate \(Pos5).
- 9.3. Bravo will let the ethanol solution and the beads incubate for 30 seconds before aspirating all of the liquid from the wells \(Pos5) and dispensing the waste fluid at Pos9.
- 9.4. Bravo will then perform a second identical ethanol wash.
- 9.5. After the waste from the 2nd ethanol wash is dispensed at Pos9, set a timer for one minute. The tips will then be returned to Pos2.
- 10. When the timer alarms, cover the plate \(Pos5) and let the plate dry for two additional minutes.
Note: Before continuing, inspect the reaction plate to make sure that all of the traces of ethanol are evaporated. The beads should be completely dry but not cracked. Make sure the beads are not over-dried as that reduces DNA recovery.
- 11. Next, Bravo will prompt you to move the DNA plate from Pos5 to Pos4 and to place the QEB plate at Pos6. Do not move the DNA plate yet, but place the QEB plate at Pos6. Remove the cover on the QEB plate and click ‘Continue’.
- 12. The program will then add 25 uL QEB \(Pos6) to the DNA plate \(Pos4) and incubate for a minimum of four minutes.
- 12.1. Bravo will retrieve tips from the tip box at Pos1 and aspirate QEB from Pos6.
- 12.2. Bravo will then dispense the QEB into the DNA plate at Pos4. Pause the protocol when the machine hovers above Pos4. Uncover the DNA plate \(Pos5) and move it to Pos4.
- 12.3. The tips will then be returned to Pos1.
- 12.4. Cover the DNA plate \(Pos4) when the machine is no longer in motion.
- 13. While waiting for the protocol, prepare the PCR mastermix in a tube and aliquot into one row of a new 96-well plate. Label this the “reservoir” plate.
- 14. Prepare the PCR brew in a 5 mL tube according to the following calculator:
- 15. Aliquot 211 uL of the mix into one 12-well strip tube.
- 16. Change the tip box at position 2 on Bravo. Pause the program and update the tip editor with Box-position2.
- 17. Place the master mix strip-tube on row A of a chilled cool-rack at position 7 on Bravo.
- 18. Place a LoBind plate on a chilled cool-rack at position 6 on Bravo.
- 19. Place the elution plate back on the Bravo deck: on top of the magnet in position 5.
- 20. The robot will first aliquot the master mix brew from the 12-well strip tube into the new LoBind plate 23 uL/well at position 6. Then it will transfer \(Pos5) the elute DNA 25 uL/well into the brew plate \(Pos6) and mix.
- 21. Using a P20 multichannel pipette, manually add 2 uL of the index primer from the Indexing Primer plate to each reaction. Label this plate “PCR.”
- 22. Spin down the plate at 3000rpm, 4C, for 2min.
- 23. Incubate the “PCR” plate in the thermocycler using the program pcr-gsc using 7 cycles. Check the cycle number and setting before starting PCR.
pcr-gsc program:
98°C for 1 minute
98°C for 30 seconds
65°C for 15 seconds
70°C for 15 seconds
70°C for 5 minutes
4°C forever
- Bead cleanup after PCR
- 1. Aliquot 50 uL of beads into each well of the “beads” plate. Do NOT use a reservoir for this step as the beads will settle and the PEG concentration will not be consistent.
- 2. Set up the bravo exactly as stated in Bead-Cleanup and End-Repair \(step 7) except with the following parameters:
DNA volume = 50 uL
Bead volume = 45 uL
Elution volume = 27 uL
- 3. The program will add the beads \(Pos6) to the DNA plate \(Pos4).
- 3.1. Bravo will retrieve tips from Pos3 and transfer 50uL of beads from Pos6 to the DNA plate at Pos4 and mix. The tips will then be returned to Pos3.
- 3.2. Pause the protocol and cover the DNA plate.
- 4. The program will aliquot the ethanol into the 96 well reservoir \(Pos9àPos8)
- 4.1. Bravo will retrieve tips from the tip box at Pos2 and aliquot ethanol from the reservoir \(Pos9) into the ethanol plate \(Pos8). Pause the program when the machine hovers above Pos9 to pour the fresh 70% Ethanol into the reservoir. The tips will be returned to Pos2 after the last of the ethanol is aliquoted.
- 4.2. Pause the program and cover the ethanol plate at Pos8.
- 5. The program will then incubate the DNA plate with the beads for 8 minutes \(10 minutes total).
- 6. After incubation, the program will prompt you to put the plate on the magnet and replace the ethanol reservoir from Pos9 with an empty Axygen 450 waste plate.
- 6.1. Place the plate on the magnet at Pos5 and incubate for two minutes \(set a timer).
- 6.2. Click ‘Continue’ when the timer alarms and incubate for an additional 3.5 minutes.
Note: Check the walls of the wells to ensure that all of the liquid is at the bottom of the well before placing the plate on the magnet. Spin for 3-4s in the centrifuge if this condition is not met.
- 7. Next, Bravo will remove the waste fluid from the DNA plate \(Pos5).
- 7.1. Bravo will retrieve tips from the tip box at Pos3 and proceed to remove all of the liquid from the DNA plate at Pos5. Pause the protocol when Bravo hovers above Pos5 and uncover the plate.
- 7.2. Bravo will dispense the waste fluid at Pos9. The tips will then be returned to Pos3.
- 8. Next, Bravo will perform two 70% ethanol washes.
- 8.1. Bravo will retrieve tips from the tip box at Pos2 and then aspirate 70% ethanol from Pos8. Pause the protocol when the Bravo machine hovers above Pos8 to remove the cover on the ethanol plate \(Pos8).
- 8.2. The ethanol will be gently dispensed into the DNA plate \(Pos5).
- 8.3. Bravo will let the ethanol solution and the beads incubate for 30 seconds before aspirating all of the liquid from the wells \(Pos5) and dispensing the waste fluid at Pos9.
- 8.4. Bravo will then perform a second identical ethanol wash.
- 8.5. After the waste from the 2nd ethanol wash is dispensed at Pos9, set a timer for one minute. The tips will then be returned to Pos2.
- 9. When the timer alarms, cover the plate \(Pos5) and let the plate dry for two additional minutes.
Note: Before continuing, inspect the reaction plate to make sure that all of the traces of ethanol are evaporated. The beads should be completely dry but not cracked. Make sure the beads are not over-dried as that reduces DNA recovery.
- 10. Next, Bravo will prompt you to move the DNA plate from Pos5 to Pos4 and to place the QEB plate at Pos6. Do not move the DNA plate yet, but place the QEB plate at Pos6. Remove the cover on the QEB plate and click ‘Continue’.
- 11. The program will then add 27 uL QEB \(Pos6) to the DNA plate \(Pos4) and incubate for a minimum of four minutes.
- 11.1. Bravo will retrieve tips from the tip box at Pos1 and aspirate QEB from Pos6.
- 11.2. Bravo will then dispense the QEB into the DNA plate at Pos4. Pause the protocol when the machine hovers above Pos4. Uncover the DNA plate \(Pos5) and move it to Pos4.
- 11.3. The tips will then be returned to Pos1.
- 11.4. While the DNA is eluting, cover the DNA plate \(Pos4) when the machine is no longer in motion.
- 11.5. After the incubation for the DNA elution is over, spin the plate for 10-20 seconds in plate centrifuge.
- 11.6. Place the plate on the magnet \(Pos5) and uncover the plate.
- 12. Place a new \(labelled) LoBind plate at Pos6. Bravo should elute the supernatant directly into new plate.
- 13. Cover the plate with a plastic cover, spin down at 2000g for 1min, 4°C. Proceed to the next step or seal with an aluminum cover and store at -20˚C.
- qPCR QC
- 1. Work at post-PCR bench. Perform all work on ice.
- 2. Place a 384 well coolrack in -20˚C freezer to chill. Get fresh ice.
- 3. Turn on ViiA7 and it’s computer. Open the program ViiA 7 1.2.
- 4. Wipe down and turn on Bravo. Make sure it is 96ST head. Load VWorks as described previously. Choose program: PBAL-qPCR pro.
- 5. Label a clean 96 well plate as a “reservoir”. Prepare two 1.5ml amber vials for qPCR brew on ice. Label one as “target”, the other one as “library”.
- 6. Prepare the brews according to the following calculators.
- 7. Aliquot 76 uL/well of the target brew into one row of reservoir plate, label the row “target”.
- 8. Aliquot 76 uL/well of the library brew into second row of reservoir plate, label the row “library”.
- 9. Retrieve qPCR-384 well plate. Mark A1 at A1 corner. Mark on the left of wells the rows for target reactions, use quadrant A. Mark on the right of the wells the rows for library reactions, use quadrant 4.
- 10. Working on ice, aliquot 9 uL of brew into each well as defined from reservoir.
- 11. Add negative qPCR control wells \(9 uL of brew) manually to the bottom right corner of the plate.
- 11.1. Negative control for target: O22 and O24
- 11.2. Negative control for library: P21 and P23.
- 12. Cover the qPCR plate and keep it on ice. Keep the plate protected from light.
- 13. Pour EB buffer \(post-PCR) into a reservoir and using P200 multichannel aliquot 100uL/well into a 96 well plate and cover with a plastic seal.
- 14. Set up Bravo deck as per image below:
Position 1: empty 384 tip box
Position 2: empty
Position 3: new box of 384 tip box
Position 4: coolrack + DNA sample plate
Position 5: chilled coolrack + 384 qPCR plate with brew
Position 6: coolrack + EB buffer plate
Position 7, 8, 9, empty
- 15. Click “Run protocol”. VWorks will prompt you to configure the tip state editor.
- 16. Select Pause, click on Tools, then Tip State Editor. Click “…” button on the right of the tip box name and adjust the tip state to match the tip boxes on the Bravo deck. Repeat for all tip boxes.
Hit Continue and start the Bravo process.
- 17. The Bravo will add the DNA into the target wells and dilute the DNA 1:100 in EB buffer before transferring into library wells.
- 18. After Bravo program is done remove the plate from the Bravo deck.
- 19. Seal the plate very well with a MicroAmp optical qPCR cover. Use the MicroAmp Adhesive Film Applicator to create a good seal, especially on the perimeter of the plate.
- 20. Spin the plate down at 3000 rpm, for 1 min, at 4°C. Place the plate inside the ViiA7 instrument.
Make sure that A1-well matches the A1 position on the tray.
- 21. In the ViiA 7 software click “New experiment”. Set up plate. After set up save it as template file **.edt for later use.
Target will be quadrant 1, and Library will be quadrant 4. \(e.g. figure below)
Negative control for target: O22 and O24
Negative control for library: P21 and P23
- 22. After set up the template, check that the cycle settings are correct:
98°C for 1min
98°C for 15 sec
60°C for 30 sec
72°C for 30 sec
- 23. The reaction volume should be 10uL and the passive reference should be set to none.
- 24. Save qPCR run to your folder, name it with plate label and date.
- 25. Hit “Run” button, the qPCR will start. Leave qPCR running overnight or if possible, wait for the qPCR results. If you wait for the qPCR, complete the pooling and ethanol precipitation step so it can be incubated at -20oC for overnight.
- 26. Clean up your workstation. Remove garbage. This concludes Day 3 of this protocol.
DAY 4
- qPCR Analysis, sample pooling and precipitation
- 1. Analyze qPCR data using qPCR pipeline.Rmd following instructions.
# 1. Change the title to desired file name
# 2. Select the correct excel file \(press tab betwene quotes, choose from the list)
# 3. Make sure the wells are defined \(positive, negative, tencell,hundredcell)
# 4. Set cutoff to be between 8-10
# 5. Press "Knit HTML"
# 6. View the plot, if needed, adjust cut off value slightly
# 7. Press "Knit HTML" again
# 8. Check through figures, save/print results and confirm correct file names
# 9. Upload to JIRA, under the appropriate library construction ticket
\`\`\`
- 1.2. Define a cutoff for success
Successful passed samples will cluster well in plot figure, the cutoff should be set right above the clustered plots. For example:
- 1.3. Check that there are no systematic errors with the plate
- 2. Percentage of passed samples should be above 75-80%. Check that the average CT values of the passed wells is consistent with previous sample plates \(Target and Library).
Note: Average CT value for Target should be between 21-23, average CT value for Library should be between 13-15. Delta CT between Target_Ct and Library_Ct should be between 6-9.
- 3. Working on ice, pool all of the passed wells and negative controls together into a 2mL tube. Exclude failed wells and positive controls \(usually G1 or as marked).
- 4. Measure the total volume and, working on ice, set up ethanol precipitation as follows:
X \(volume pooled in uL)
0.1*X of 3M Sodium Acetate pH 5.5 \(in uL)
- 5. Mix well by pulse-vortexing. Add 1.3 uL mussel glycogen \(marked as MG, 20mg/mL) and mix well by pulse-vortexing.
- 6. Calculate the total volume and add 2.5X\(total volume in uL) of cold 100% ethanol. Vortex thoroughly. It is important that the solution is very well mixed. Split the volume across multiple 1.5mL tubes.
- 7. Incubate precipitations at -20˚C for at least 2 hour but overnight is preferred.
- 8. While waiting, pre-chill the centrifuge to 4˚C.
- 9. After -20˚C incubation, place all tubes in the same orientation in the pre-chilled to 4°C centrifuge.
- 10. Spin down at 15,000g \(max rpm) at 4°C, for 40 min.
- 11. Prepare fresh 70% ethanol using ultrapure water. Take out and aliquot of 20% PEG SeraMag beads to warm up to room temperature for at least 30 min.
Note: The following steps must be performed on ice.
- 12. After centrifugation visually locate the pellet. Carefully discard the supernatant while avoiding the DNA pellet.
- 13. Add 1mL of 70% ethanol, plate the tube inside the centrifuge in the same orientation as before, and spin down at 15000g \(max rpm) at 4°C, for 4 min.
- 14. After centrifugation visually locate the pellet and carefully aspirate the supernatant.
- 15. Spin down the tube in minifuge to collect all of the ethanol on the bottom. With P10 remove the final traces of ethanol.
- 16. Dry the pellet at room temperature until the pellet becomes translucent \(maximum 5 minutes).
Keep a close eye on the pellets so that they do not over-dry.
- 17. When the pellet becomes clear, resuspend the DNA in each tube with 20 uL of room temperature EB buffer. Mix up and down with the pipette.
- 18. After the pellets are fully dissolved, pool all volume into one tube. Measure the volume and top up with EB buffer to a multiple of 50uL. For example, if the volume is 80uL add an additional 20uL of EB buffer to a total volume 100uL \(which is 2X 50uL).
- Library pool purification using SeraMag beads
- 1. Make sure that the 20% SeraMag beads have fully reached room temperature before proceeding and use freshly made \(on the same day) 70% ethanol.
- 2. Working at post PCR work station, using a single channel P100, aliquot the pooled and precipitated single cell PCR library products - 50uL per well into one row of a new LoBind plate.
- 3. Mix the 20% SeraMag beads very well and aliquot 55ul per well into one row of a reservoir plate \(corresponding to the number of DNA wells). Using P200 multichannel, to each 50uL of DNA add 45uL of room temperature 20%PEG SeraMag beads \(0.9X ratio) and immediately mix by pipetting up and down at least 10X.
- 4. Incubate at room temperature for 10 min.
- 5. After 10min incubation move the plate to the magnet and incubate for 3 min. Make sure that all of the beads are separated to the sides and the solution is completely clear before proceeding.
- 6. Keep the plate on the magnet for the next few steps.
- 7. Without disturbing the beads, using a single channel P200, carefully remove the clear supernatant. Make sure not to remove any beads.
- 8. On the magnet, using multichannel P200, add 150ul of 70% ethanol and incubate for 30 seconds.
- 9. From each well, carefully aspirate the ethanol using single channel P200.
- 10. Using multichannel P200, add 150uL 70% ethanol and incubate for 30 seconds.
- 11. From each well, using single channel P200 set to 170uL, carefully remove all of the ethanol.
- 12. After the second wash keep the plate still on the magnet. Using P10 aspirate the remaining traces of ethanol from the bottom of each well. Make sure not to remove any beads,
- 13. Let the beads dry at room temperature for 2 min. Do not over-dry the beads. You should not see any cracks.
- 14. When the beads are dry, remove the plate from the magnet. Using P20, add 15uL of EB buffer and mix very well by pipetting up and down. Make sure that all of the beads are re-suspended. The solution should be uniformly brown.
- 15. Incubate at room temperature for 3min.
- 16. After 3min elution, place the plate back onto the magnet and incubate for 1 min. Make sure that all of the beads are pulled to the sides and the solution is completely clear.
- 17. After 1min incubation, using P20 carefully transfer the supernatant \(containing the eluted DNA) into a new 1.5 mL tube. Label the tube with sample/pool ID.
- 18. Use 1uL of the sample for Qubit assay and run another 1uL on HS Agilent to observe the fragment distribution. Expected yield should be > 1 ng/uL. Average size of the final product should be around 300bp.
- 19. Store the sample at -20C until ready for sequencing. Record all of the data in your notebook.
- 20. Clean up your workstation. Remove garbage. This concludes Day 4 of this protocol.