Cloning from PCR

[pipette_man]

Hi,
unfortunately I have begun cloning from a PCR using Klenow and then blunt end ligation.

Any tips for conducting the PCR cloning in a better way next time?
thanx

[admin]

Hello pipette_man,
hope you had a good year

I hate klenow/blunt end ligations.
I would try to subclone next time into a TOPO vector, if you can convince your supervisor that its worth the price.

It is a bit pricey, however TOPO TA kits save some time and effort in cloning pcr fragments.
What I would do is either PCR the fragment with primers that have your restriction fragment ends of interest OR insert into a suitable TOPO vector that has your restriction sites in its MCS (multiple cloning site).

Cheers!

[Rene]

we usually do it with primers containing the restriction site, I think it's cheaper than using TOPO

[serenacorallini]

I usually clone an insert by PCR using primers with restriction site but sometimes I have problem with PCR (no band) is it possible that little tail add to primer decreases the yield of PCR product?