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

File:08202014.JPG

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.

File:ZhangLab 2 2014-08-20 18hr 19min.jpg