restriction digestion of PCR product? - (Jun/19/2006 )
Hi,
I'm having a terribly hard time cloning a PCR product in an expression vector.
I'm using primers containing RE sites in order to generate a PCR product with cohesive ends and paste the digested PCR product directly into the plasmid, trying in this way to skip the TA-cloning step.
PCR works ok but I never get recombinant-positive trasformants, only re-circularised vectors.
I thought saving money on TA cloning was a smart idea but, since I've been trying so hard and for so long, I'm starting to think that I'd better have bought it and used it since day one....
Any comment or suggestion on that?
Thanks
are you using the same site on each end of your product? if so, perhaps you'd like to try directional cloning or CIP treat your vector?
Could u tell us your digestion protocol along with the sites u r using as incomplete digestion usually results in recircularised vectors.
i think the problem came for your vector and/or your ligation step.
Tip : heat at 65° the mix insert + plasmid and cool gently on the bench before adding enzyme and buffer.
Another problem can be cutting the PCR fragment when Taq is still present. The enzyme will extend the 3' end of the cut site, preventing sticky end ligation. You have to remove the Taq and dNTPs before cutting with enzymes leaving a 5' overhang.
Your restriction enzymes will need 4-6 bases outside of the cut site, also, to allow the enzymes to cut properly.
No need for that (at least in theory), since I'm using two different enzimes.
Thanks
This makes sense. I digested with excess enzyme units/time, but I'll try leaving it overnight.
Thanks
Tip : heat at 65° the mix insert + plasmid and cool gently on the bench before adding enzyme and buffer.
I assume heating will help getting rid of secondary structures and other crap like that?
Nice! I'll try that. Maybe it will help in case the critical step is ligation.
Thanks
Your restriction enzymes will need 4-6 bases outside of the cut site, also, to allow the enzymes to cut properly.
I do gel purification before cutting, and I have 4 extra bases (ATAT) flanking the cut site.
Thank you
if you can avoid the gel step it's better. Generally a column is faster, roughly cheaper if we assume the purity grade of agarose is high, and due to the fact i have more success when not purifying by gel.