Arturo Casini:Protocols

PCR

2 temp steps (2 different annealing temperatures)

to amplify miniprepped plasmid DNA

mix in small 0.2 ml thin walled PCR strip tubes

mix in this order

Distilled H20 18.75 ul

Barnes buffer (B'S on the tube) 2.5 ul

Forward primer (100 ng/ul) 1 ul (4 ng / ul)

Reverse primer (100 ng/ul) 1 ul (4 ng / ul)

Template DNA (miniprep) 1 ul (0.2 ng/ul)

dNTP mix (10 mM) 0.5 ul

PfuUltra II fusion DNApol 0,25 ul

TOTAL 25 ul

1x

95 degs, 30 secs

10x

95 degs, 30 secs

annealing portion Tm -3 degs, 30 secs (if more than one, the lowest)

68 degs, 15 secs + 15 secs for each kb

20x

95 degs, 30 secs

whole primer Tm -3 degs, 30 secs (if more than one, the lowest)

68 degs, 15 secs + 15 secs for each kb

1x

68 degs, 2 mins

1x

4 degs, infinite

Cover lid temperature: 105-110 degs