TOPO TA cloning advice
Posted 17 May 2010 - 06:50 AM
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...
Posted 17 May 2010 - 07:51 AM
I never trust anything that can't be doubted.
Posted 17 May 2010 - 05:26 PM
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.
Posted 18 May 2010 - 12:49 PM
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Posted 23 May 2010 - 11:34 PM
Have a nice day!