For PCR reactions up to 50 μL add the following to each PCR Tube:
Substance
GoTaq
Q5
Phusion
Forward Primer
2.5
2.5
2.5
Reverse Primer
2.5
2.5
2.5
Polmerase
.2
25 μL mastermix
.5
dNTPs
.5
-
1
dH2O
14.3
18.0
33.5
Buffer
5
-
10
DNA
touch of colony
touch of colony
touch of colony
PCR Machine Cycling Times and Temperatures:
E. coli and Sinorhizobium Plasmids
Step Temperature Time
Initial Denaturation 95 5:00 min
30 Cycles Denaturation 95 15 sec
Annealing 58 15 sec
Extension 72 2-4 (1min per kb)
Final Extension 72 5-10 min
Load on 1% agarose gels and run electrophoresis tank until the coloured bands are near the bottom of the gel. Use U:genius machine to photograph and visualise bands.
