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
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