TOPO TA cloning advice - (May/17/2010 )
I want to clone some PCR products (amplified with Taq polymerase) using the TopoCloning kit. These PCRs are usually producing some kind of smear, so I have thought of running the whole PCR product in a gel, cut the band (2 kb), and purify it using the MinElute Gel extraction kit (it is a kit that uses columns).
However, I'm afraid that the purification will reduce the overhanging A that will be needed to clone into the TOPO vector. I must get as many positive colonies as possible, so it is very important for me not to disturb the overhanging A.
Can you give me some advice for this? Do you have any experience in topocloning after gel purificacion?
And another question is: Would freezing be worse than gel purificacion? because if it is not worse, I could do the gel purification by freeze and squeeze instead...
We gel-purified the products regulary with MinElute kit and had no problems with TOPO TA cloning. I would say that repeated freeze/thaw is worse for the A overhangs.
A single freeze-thaw cycle should be OK, it is repeated ones that do the damage.
If you are looking to clone a specific sequence use a proof reading polymerase such as pfu, KOD or Expand and add A overhangs by adding Taq and dATP after the amplification.
You may be able to do this for your products anyway, if you are worried about loosing the A's.
try to eliminate the cause of the smear so you don/t have to do gel purification. Check that you are no adding too much template to the pcr reaction and that the template isn't degrade. I used to clone with topo ta and never purified the pcr, just add the pcr to the vector for ligation etc,. For that you need a nice, sharp, one product band.
Thank you all for your answers and recommendations!
Have a nice day!