Daniel:Notebook/PosSequencing/2017-7-7
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emPCR8[edit]
Protocol-Part 2[edit]
- Emulsion Breaking
- With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip
- Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube
- Vortex the pooled sample in the 1.5 mL tube for 30 seconds
- Spin down the sample 13,000 rpm for 5 minutes
- Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL ethyl acetate and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove ethyl acetate without disturbing the pellet
- Add 1000 uL diethyl ether and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove DE without disturbing the aqueous phase
- Allow DE to dry in the fume hood; resuspend beads in the solution and take 15 uL aside for testing
- Phosphorylation
- Pull down beads, remove supernatant, and wash with 100 uL DynaT-buffer (Dynabuffer + 0.05% Tween20)
- Pull down beads and remove supernatant
- Resuspend the beads in the following mixture
- Resuspend each sample in 49 uL master mix
- Add 1 uL T4 PNK to each
- Incubate for 30 minutes at 37C
- Heat inactivate by incubating for 20 minutes at 65C
- Lambda Exonuclease Digestion
- Pull down with magnet and remove supernatant
- Make the following master mix
- 16 uL 10X Lambda exonuclease reaction buffer
- 140.8 uL nfH2O
- Resuspend beads in 49 uL master mix
- Add 1 uL lambda exonuclease (10 U/uL)
- Incubate for 30 minutes at 37C
- Heat kill enzyme by incubating for 10 minutes at 80C
- Magnet pulldown, and resuspend in 50 uL 2XSSC
- Dye hybridization
- Aliquot 20 uL sample to a new tube
- Add 20 uL 4X SSC and 50% formamide
- Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
- Incubate in the dark (RT) for 15 minutes
- Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
- Add 15 uL to a slide for analysis
- Seal coverslip with nail polish and wait approximately 30 minutes to dry
- Imaging Parameters
- Confocal Microscope
- 488 Laser-3.00%
- PMT Trans-Gain 225V
- PMT1-Range:500-550-Gain 550V
- PMT3-Range:750-800-Gain 600V
- 642 Laser-8.00%
- PMT Trans-Gain 300V
- PMT1-Range:500-550-Gain 6000V
- PMT3-Range:750-800-Gain 550V
Reagent | uL 1X Reaction | uL 3.2X Master Mix |
T4 PNK Buffer (10X) | 5 | 16 |
ATP (10 mM) | 5 | 16 |
T4 PNK (10 U/uL) | 1 | 3.2 |
nfH2O | 39 | 124.8 |
Total | 50 | 160 |