User:Anthony Salvagno/Notebook/Research/2009/09/24/Another PCR of the anchor
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.
Comparison
- 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
Only tubes 5 and e worked. Lanes:
- 1x ladder
- 2x ladder
- 3x ladder
- 5x ladder
- tube 1
- 2
- 3
- 4
- tube 5
- tube a
- b
- c
- d
- 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.
