colony pcr - (Jul/27/2009 )

hi guys, just wondering...

in colony pcr is it possible that the set of primers replicate the entire sequence of recombinant plasmid dna?

eg. plasmid is 6 kb
for - rev primers is around 400bp

is it possible to get a product of 6.4kb as a product of colony pcr?

If you did not use a super efficient polymerase I think it is impossible. I do not know any enzymes that can extend that long

That is possible.

I have a plasmid containing an insert, and the total size is 7kb. I wanted to do a deletion PCR to delete a 200+bp region so I basically designed a forward and reverse primer flanking the region to be deleted and carried out the PCR. Did it with PfuTurbo and I got my PCR products, which I ligated and transformed into E.coli and sequenced them. Got the right clone that I wanted.

orangee on Jul 27 2009, 07:02 AM said:

Yes, it's possible, but if you're doing a PCR for a 400 bp product, you shouldn't be extending for long enough to get a 6.4 kb product.

orangee on Jul 27 2009, 08:02 PM said:

I think it is impossible:

1.Iit needs several minutes to extend a segment of 6.4kb;
2.The enzyme can not extend DNA in a direction from 3' to 5' , therefore, when the direction of your primers are confirmed, the product is confirmed.