AlanFung:LabNotes/CTCF/2011-8-19: Difference between revisions
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>Alan6017518 |
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**M13 Reverse | **M13 Reverse | ||
**CAGGAAACAGCTATGAC | **CAGGAAACAGCTATGAC | ||
**Invitrogen M13R | |||
**CAGGAAACAGCTATGAC | |||
**M13 Forward | **M13 Forward | ||
**GACCGGCAGCAAAATG | **GACCGGCAGCAAAATG | ||
**Invitrogen M13F | |||
**GTAAAACGACGGCCAG | |||
*Order for M13F was reversed |
Revision as of 00:07, 25 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
- Either the polymerase or the pcr condition is not optimised, I will test both parameters with the control reactions to test the activity of the polymerase
Producing the Control PCR Product
Content | Volume per Reaction |
Control DNA template (100ng) | 1 |
M13 F+R (2.5uM each) | 8 |
Sterile water | 16 |
TagMM | 25 |
Total Volume | 50 |
94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever
Page Gel Analysis
File:ZhangLab 2 2011-08-23 18hr 26min.jpg
Results
- No amplicons seen on page gel, I guess the polymerase has gone bad, will repeat with a new tube of taq polymerase
- I double checked the condition for PCR and there are no obvious error.
- Will repeat experiment with Invitrogen supplied primers, new tube of NEX Taq2X mastermix with the control DNA
Troubleshooting
Content | Volume per reaction | Content | Volume per reaction |
Control DNA Template (100ng) | 1 | Control DNA Template (100ng) | 1 |
Invitrogen:M13-R | 2 | M13 F+R (2.5uM each) | 8 |
Invitrogen:M13-F | 2 | Sterile Water | 16 |
Sterile Water | 20 | NEB Taq2X MM | 25 |
NEB Taq2X MM | 25 | Total | 50 |
Total | 50 |
94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever
- Appears that the polymerase is not the problem, but the primers.
- Check the primers ordered
- Sequence ordered
- M13 Reverse
- CAGGAAACAGCTATGAC
- Invitrogen M13R
- CAGGAAACAGCTATGAC
- M13 Forward
- GACCGGCAGCAAAATG
- Invitrogen M13F
- GTAAAACGACGGCCAG
- Order for M13F was reversed