AlanFung:LabNotes/2014/2014-8-20
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- Continue from 8/14/2014 [[1]]
Beads Purification[edit]
- Vortex to resuspend beads
- Add 62.5ul of beads to 50ul amplicon (1.25X)
- Mix by pipetting up and down & wait 15min
- transfer to magnet and let it sit for 5min (M)
- Discard supernatant & wait for 10min (M)
- Wash w 160ul 75% ETOH & wait for 1min (M)
- Discard ETOH and repeat wash (M)
- Let dry for 10min (M)
- Remove from magnet
- Resuspend w 42ul EB buffer & wait for 2min
- Transfer to magnet & wait for 5min (M)
- Transfer purified amplicons to a clean tube (M)
qPCR Test[edit]
Components | Conc | unit | Final conc. | unit | Volume (ul) | 6 rxn mix |
Purified 1st round DNA | 3 | 0 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 2 | 12 |
dNTP mix | 10 | mM | 0.25 | mM | 0.25 | 1.5 |
TruS_F/R | 10 | uM | 0.3 | uM | 0.3 | 1.8 |
50X SYBR | 50 | X | 0.4 | X | 0.08 | 0.48 |
Phusion HF | 2 | unit/ul | 0.1 | 0.6 | ||
H2O | 4.27 | 25.62 | ||||
Total | 10 | 42 |
- Aliquot 7ul + 3ul DNA template
- Mix well
- I will be running 7-8 cycles
Test_Round_scRRBS_Amp 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 7 and 8 cycles --> 72C for 2min
PAGE Gel Analysis[edit]
- Added 2ul 6X loading dye to each strip tube & mix well
- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min.