AlanFung:LabNotes/CTCF/2011-8-19: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 28: Line 28:
==Results==
==Results==
*For some reason, no amplicons were formed, I suspect the ligation failed
*For some reason, no amplicons were formed, I suspect the ligation failed
==Troubleshoot==

Revision as of 18:21, 23 August 2011

Preparation for Sanger Sequencing

Objective

  • Clones grew well after overnight incubation, prepare clones for sanger sequencing. (a bit overgrown and most of the clones are blue in color)

Protocol

PCR amplification and preparation for sanger sequencing

  • Add 12.5ul of 100ul of F and R primer and add 475ul sterile h2o to make 2.5uM primer mix
Content Volume per reaction MM
DNA Pick one colony per well Do not add to MM
H2O 25 205
M13-21 F+R Primer (2.5uM each) 8 65.6
2X Taq MM 15 123
Total 48 393.6
  • Add 48ul MM to each colony
94C 10 min to break up the cells
94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever

PAGE

File:ZhangLab 2 2011-08-19 16hr 58min.jpg

Results

  • For some reason, no amplicons were formed, I suspect the ligation failed

Troubleshoot