AlanFung:LabNotes/CTCF/2011-8-19: Difference between revisions

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>Alan6017518
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*I double checked the condition for PCR and there are no obvious error.
*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
*Will repeat experiment with Invitrogen supplied primers, new tube of NEX Taq2X mastermix with the control DNA
==Troubleshooting==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Content'''
| align="center" style="background:#f0f0f0;"|'''Volume per reaction'''
|-
| Control DNA Template (100ng)||1
|-
| Invitrogen:M13-R||2
|-
| Invitrogen:M13-F||2
|-
| Sterile Water||20
|-
| NEB Taq2X MM||25
|-
| Total||50
|}

Revision as of 21:05, 24 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
Control DNA Template (100ng) 1
Invitrogen:M13-R 2
Invitrogen:M13-F 2
Sterile Water 20
NEB Taq2X MM 25
Total 50