Pfx polymerase for site-directed mutagenesis - (Nov/14/2006 )
Hi all,
Has anyone here ever used Pfx polymerase (invitrogen) for PCR based site-directed mutagenesis before please? If yes, and you have got it worked, could you please be so kind to leave the protocol?
Thanks a lot!!!!!
-flametree-
Haven't used it, but I would try the quickchange method. For longer plasmids I've compared PfuUltra with pfx (just the PCR) and pfx couldn't amplify my 8 kb, it gave me (for other pcr's) higher yields than pfu (up to about 3kb). Haven't compared fidelity.
Invitrogen actually also has a site directed mutagenesis method on their website, calles genetailor, you might wanna check it out: https://catalog.invitrogen.com/index.cfm?fu...European%20Main
-vairus-
QUOTE (vairus @ Nov 15 2006, 08:57 AM)
Haven't used it, but I would try the quickchange method. For longer plasmids I've compared PfuUltra with pfx (just the PCR) and pfx couldn't amplify my 8 kb, it gave me (for other pcr's) higher yields than pfu (up to about 3kb). Haven't compared fidelity.
Invitrogen actually also has a site directed mutagenesis method on their website, calles genetailor, you might wanna check it out: https://catalog.invitrogen.com/index.cfm?fu...European%20Main
Invitrogen actually also has a site directed mutagenesis method on their website, calles genetailor, you might wanna check it out: https://catalog.invitrogen.com/index.cfm?fu...European%20Main
Thanks a lot! I just used up the Pfu turbo but I've got loads of Pfx. That's why I was thinking to use Pfx instead of Pfu turbo. However the Pfx seems gave me only a big smear but nothing. I think I probably still have to order some more Pfu.
-flametree-