4 replies to this topic

[vgupta]

Has anyone carried site directed mutagenesis of a 20 kb plasmid? I have tried it using Quick change kit according to their protocol but it is not working. I don't get any colonies after transformation. The extension time I am using is 1 min/kb. I have tried 2 min/kb also with the addition of extra enzyme after 7 cycles. I were using 1 mM dNTPs. With 2 min extension time I get a smear and with 1 min I don't get anything. With less dNTPs (500 uM) and at annealing temperature of 68 deg instead of 60 I get multiple bands which are smaller than 20 kb . Does any one have any suggestions or conditions for carrying out that experiment?
Thanks.

[vgupta]

No one?

[KateGUR]

I have also been trying to make a specific mutation in a plasmid of 20Kb.  I have tried Statagene's Lightning kit, but up to now, no clones!  This is only the second time I tried, and I have gotten several clones growing, but all do not have the mutation!  I'd be interested in any help, or other's experiences!

[mastermi]

Don't you have a possibility to carry out the site-directed mutagenesis on a smaller plasmid and clone the fragment into the 20 kb plasmid?
I'm always using plasmids with a size of 2-4 kb for s-d mutagenesis, once tried it with a 6 kb one, and that already didn't work out...

[T C]

Mastermi is right.  Thats the way to go about it. What I do is to subclone a region that covers the site to be mutated, mutate the subclone and insert the fragment back.  Use unique sites to take it out.  However, I have gone upto 8 kb but don't prefer it as you are not sure of errors introduced during PCRing out long fragments.

Best,
TC

mastermi, on Apr 17 2009, 10:52 PM, said:

Don't you have a possibility to carry out the site-directed mutagenesis on a smaller plasmid and clone the fragment into the 20 kb plasmid?
I'm always using plasmids with a size of 2-4 kb for s-d mutagenesis, once tried it with a 6 kb one, and that already didn't work out...