AlanFung:LabNotes/CTCF/2011-8-19: Difference between revisions
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>Alan6017518 (Created page with '==Preparation for Sanger Sequencing== ===Objective=== *Clones grew well after overnight incubation, prepare clones for sanger sequencing. ==Protocol== ===PCR amplification and p…') |
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===PCR amplification and preparation for sanger sequencing=== | ===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 | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Content''' | | align="center" style="background:#f0f0f0;"|'''Content''' |
Revision as of 20:48, 19 August 2011
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) | 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