Alice:LabNotes/2009-9-1
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Re-amplify and size selection the shotgun libraries from 8-26-09[edit]
- detailed library construction can be found under LabNotes 8-28-09
PCR amplification[edit]
DNA(libraries from 8/26/09) 1 (DF and foreskin) PCR_up (100uM) 0.2 PCR_lo (100uM) 0.2 2x Phusion GC master mix 50 50x SYBR Green I (Invitrogen) 0.2 dH2O 40
98C 1min -> 5 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min
Size selection and purification of the constructed sequencing libraries[edit]
- The size range of the library DNA is ~175-225 bp. File:ZhangLab 2 2009-09-01 14hr 47min.jpg
QPCR[edit]
DNA 1 Syb_FP5(100uM) 0.2 Syb_RP7(100uM) 0.2 2x Phusion GC master mix 25 50x SYBR Green I (Invitrogen) 0.2 dH2O 25 split every 50ul rxn into 2 wells, so total rxn vol for PCR setup is 50ul 98C 30sec -> 11-20 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min
qPCR result: DF6-9-9: 1nM foreskin: 1nM