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.

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:
 * 1) 1x ladder
 * 2) 2x ladder
 * 3) 3x ladder
 * 4) 5x ladder
 * 5) tube 1
 * 2
 * 3
 * 4
 * 1) tube 5
 * 2) tube a
 * b
 * c
 * d
 * 1) tube e

Koch PCR
We changed the tube reader to Sim Tube. Used a-e protocol from earlier.