AlanFung:LabNotes/CTCF/2011-8-19

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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