a kewl spreadsheet that helps with mutagenic and regular primer design
ingredients
For each 50ul reaction, in a .5ul microfuge tube:
template dna - ~100-200ng
primers - ~900 ng of each
dNTP's - 5ul of 2.5mM
polymerase buffer - dilute to proper concentration (if 10X then 5ul)
polymerase - ~5 units
MgCl2 - 3ul of 25mM
ddH2O - to 50ul
program
94C - 3 min
start cycle, 40X
94C - 1 min
40 to 55C - 1 min
72C - 1 min
end cycle
72C - 10 min
The annealing temperature (40 to 55C) should be varied on a small scale PCR before deciding on a specific temperature for the large scale.
Check your product (~5ul) on a agarose gel. For best results, use QIAGEN gel purification kit to purify.
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