AlanFung:LabNotes/CTCF/2011-8-19: Difference between revisions
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==Preparation for Sanger Sequencing== | ==Preparation for Sanger Sequencing== | ||
===Objective=== | ===Objective=== | ||
*Clones grew well after overnight incubation, prepare clones for sanger sequencing. (a bit overgrown and most of the clones are blue in color) | *Clones grew well after overnight incubation, prepare clones for sanger sequencing. (a bit overgrown and most of the clones are blue in color, we can either plate a smaller volume of tranformation mixture or dilute 10 fold with soc before plating) | ||
==Protocol== | ==Protocol== | ||
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|} | |} | ||
94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever | 94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever | ||
[[File:ZhangLab_2 2011-08-24 16hr 48min.jpg|300px]] | |||
*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, will discard the forward primer and use the ones from the kit for now. | |||
*In order to avoid having to many transformations and well-spaced colonies on the LB plates in the future I should spread 10 to 100ul from each transformation vial on LB plates, for plating smaller volumes, add 20ul soc to ensure even spreading. | |||
*It will be best to measure the concentration of the purified PCR product and purified ligation product | |||
*Make sure to calculate the amount of pcr product to use with this equation x ng insert =(10*Y bp pcr product*25ng linearized pcr blunt)/3500bp pcr blunt |
Latest revision as of 20:43, 25 August 2011
Preparation for Sanger Sequencing[edit]
Objective[edit]
- Clones grew well after overnight incubation, prepare clones for sanger sequencing. (a bit overgrown and most of the clones are blue in color, we can either plate a smaller volume of tranformation mixture or dilute 10 fold with soc before plating)
Protocol[edit]
PCR amplification and preparation for sanger sequencing[edit]
- 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[edit]
File:ZhangLab 2 2011-08-19 16hr 58min.jpg
Results[edit]
- For some reason, no amplicons were formed, I suspect the ligation failed
Troubleshoot[edit]
- 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[edit]
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[edit]
File:ZhangLab 2 2011-08-23 18hr 26min.jpg
Results[edit]
- 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[edit]
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
File:ZhangLab 2 2011-08-24 16hr 48min.jpg
- 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, will discard the forward primer and use the ones from the kit for now.
- In order to avoid having to many transformations and well-spaced colonies on the LB plates in the future I should spread 10 to 100ul from each transformation vial on LB plates, for plating smaller volumes, add 20ul soc to ensure even spreading.
- It will be best to measure the concentration of the purified PCR product and purified ligation product
- Make sure to calculate the amount of pcr product to use with this equation x ng insert =(10*Y bp pcr product*25ng linearized pcr blunt)/3500bp pcr blunt