AlanFung:LabNotes/CTCF/2011-8-19
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Preparation for Sanger Sequencing
Objective
- Clones grew well after overnight incubation, prepare clones for sanger sequencing.
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