Hal has had a revelation.
Stratagene’s QuikChange Mutagenesis Kit:Contains: PfuUltra™ High Fidelity DNA polymerase, 10X reaction buffer, dNTP mix, Dpn I restriction enzyme, QuikChange control plasmid and control primers, XL-1 supercompetent cells, pUC18 control plasmid.
I'm wondering what took him so long. I first used that method in, oh, 1996, 1997? Stratagene (patent filed in 1995) had brought out the kit, and I looked at the method and thought what is stopping me doing that with standalone reagents?
So I talked to my then-boss, ordered the kit and the individual reagents from different suppliers, and did the side-by-side comparison. Since then, I think I've ordered the kit precisely once and that was to get hold of the instruction manual, which is now available online. And made countless mutants with DpnI from NEB and bog-standard Pfu.
You can even use Taq with high fidelity if you use dNTPs near the Km . . .
Ten years, Hal. What kept you?
Comments
When I was doing my PhD everthing was made in parts/hand made. My PI hated kits. NOw i use nothing but..troubleshooting can be a real sod.
But Taq? In a mutagenesis? Dangerous ground there matey...
Posted by: Tideliar | September 1, 2006 05:46 AM
Remind me to tell you how, back in the day, I sequenced more than 3 kb (hand-poured gels) of Taq-amplified clone with no errors.
Posted by: BK | September 1, 2006 09:18 AM