Alice:LabNotes/2009-9-1

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Revision as of 16:30, 9 September 2009 by >Zsakura2 (New page: ==Re-amplify and size selection the shotgun libraries from 8-26-09== *detailed library construction can be found under LabNotes 8-28-09 ===PCR amplification=== DNA(libraries from 8/26/09)...)
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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