Daniel:Notebook/PosSequencing/2017-7-13: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 81: | Line 81: | ||
<li>Heat kill enzyme by incubating for 10 minutes at 80C</li> | <li>Heat kill enzyme by incubating for 10 minutes at 80C</li> | ||
<li>Magnet pulldown, and resuspend in 40 uL 2XSSC</li> | <li>Magnet pulldown, and resuspend in 40 uL 2XSSC</li> | ||
</ol> | </ol> | ||
</ol> | </ol> | ||
Continued [[Daniel:Notebook/PosSequencing/2017-7-14|tomorrow]] | |||
[[Category:PosSeq]] [[Category:20170712]] | [[Category:PosSeq]] [[Category:20170712]] |
Latest revision as of 16:00, 14 July 2017
emPCR 9-Concentration Comparison (Started Yesterday)[edit]
Protocol-Part 2[edit]
- Prepare Water saturated compounds
- Mix 5 mL diethyl ether and 5 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
- Mix 5 mL ethyl acetate and 5 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase
- 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 split in half for testing (40-50 uL)
- 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 1 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 40 uL 2XSSC
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 |
Continued tomorrow