User:Anthony Salvagno/Notebook/Research/2009/09/24/Another PCR of the anchor

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New Taq

Initially I had been using Taq from invitrogen, but I decided to go with NEB because each supply had 4x more Taq than the invitrogen one. I don't know if this was wise, but whatever. I am now comparing the two manuals to determine differences and to see if I can go with my old protocol.


  • NEB buffer has MgCl2 in it comes as 10x concentration
  • both have 5u/ul Taq
  • both require same amount of dNTPs

PCR Reaction

I'm going to try the new way and the old way today. I think I used too much MgCl2 according to setups I looked at in the past. I'll adjust this. {{#widget:Google Spreadsheet |key=tIKC4wLGECareZV1fXRfMFA |width=900 |height=300 }} The pipettemen are still not calibrated properly and I hope that won't fuck with everything. The reason I know this is because I should have made 525ul Master Mixes, but after pipetting 5 100ul aliquots there was well over 25ul left in the tubes (maybe like 50ul). Also I think I might have added more Reverse primer to my a-e tubes because I can't recall how I had to add it. The possibility of this is like 50%.

Gel Analysis

Capture 00019.JPG
Only tubes 5 and e worked. Lanes:

  1. 1x ladder
  2. 2x ladder
  3. 3x ladder
  4. 5x ladder
  5. tube 1
  6. 2
  7. 3
  8. 4
  9. tube 5
  10. tube a
  11. b
  12. c
  13. d
  14. tube e

Koch PCR

{{#widget:Google Spreadsheet |key=taFSMiiCjTZ2X0DaXeYpfXQ |width=500 |height=300 }} We changed the tube reader to Sim Tube. Used a-e protocol from earlier.