PCR of long fragment(approx 8 kb) - (Mar/06/2006 )

Hi,

How can we amplify a long fragment the size of which is approx 8 kb? In our lab we use the general strategy of cloning in which we first amplify short fragments then with the help of PBS we ligate them but with this we can't go for long fragments. Is there any method available to clone these long fragment directly?

Thanks

hi
i've done that with 7.6kpb. I amplify by triple master (good quality great quantity and proof reading) and cute by restriction enzymes (sites in the primers). Ligation is done with 1:6 ratio (so generally uses a lot of the PCR....

Thanks Fred. I've also tried the same thing for the same size of fragment and also got the PCR but when I did PNK and tried to ligate it in pBS it didn't work. I wanted to ask about this PNK and CIP, Should I use these or not coz for long fragment its being very difficult to clone them with the help of this strategy.

well, PNK of the insert is not necessary because restriction digest produce 5' P termini.