Will TOPO TA cloning work on "old" purified PCR product? - (Feb/12/2008 )

Hi!

Will TOPO TA cloning work on PCR product that has been gel purified and kept at -20oC for some weeks/months? Or is it advisable to use fresh PCR product (and gel purified)?

Well, the A-overhangs degrade over time. It will probably work but not very efficiently.
If you're aiming for high efficiency ligation try doing it again. If a couple of colonies are enough, just use the old PCR product.

Sometimes the poly A tails are lost with gel purification + too much time in fridge = to low cloning efficiency, so recomendation do the pcr again save time and money....

Unfortunately it seemed that the PCR is somewhat "unoptimizable" and a fresh PCR would probably require gel purification.

So I tried to blunt the old PCR product with T4 DNA Polymerase, EtOH precipitate and clone into ZERO BLUNT TOPO vector, electrotransformed and plated out 10 ul and 50 ul. No colonies grew.
Could there be something wrong with my T4 DNA Pol reaction?

Here is what I did:

Blunting:
---------
In 50 ul rxn:
PCR product (154 bp) 50 ng , 450 pmol
dNTP mix 0.1 mM each final
T4 DNA Pol (Promega) ~5 U

Incubate at 37oC for 5 min. Then phenol-chloroform-isoamylalc extraction and EtOH ppt. Resuspend in 4 ul H20 and clone into ZERO BLUNT TOPO kit as per manufacturer's protocol, using electrocompetent TOP10 cells.


(Of course as a last resort, I guess I have to go back to getting a fresh PCR.)

You could also try A tailing the existing PCR product. Do a PCR reaction with no primers, set up to run for 20 minutes at extension temperature. Use Taq, of course. Purify the product and go. This won't amplify, of course, so you'll need to add as much DNA into the reaction as you expect out. Don't overdo it, 100 ng should be more than enough. Then clone directly as you would fresh PCR product for TA cloning. If you have individual dNTPs available, you should add only dATP to the mix, but it should work with a dNTP mix.