TOPO TA Cloning - hundreds of colonies, 0 insert - (May/19/2008 )

Hello

I am having difficulty with cloning. I am trying to clone PCR product created using Taq. I am using Invitrogen's TOPO TA kit, and the transformation appears to go very well. My LB&Kanamycin plates have plenty of colonies, and overnight liquid cultures grow well also. However, when I run M13 PCR I do not get any bands. I have tried several things so far:
1) I purified the PCR product before setting up the cloning reaction
2) I have varied the amount of PCR product used
3) I have varied the amount of vector used
4) I set up the TOPO kit's control PCR reaction (PCR produced the expected 750bp band, however M13 PCR after cloning also failed to show the insert)
5) I did an extra incubation with Taq prior to cloning
6) I handed the colonies over to someone else to try M13 PCR with their own reagents
7) I used a 30 minute incubation of the PCR/vector/salt mixture prior to adding to cells (as suggested by TOPO manual)
8) I set up an EcoRI restriction digest on purified plasmid (using eppendorf fastplasmid kit), resulted in only one band

I am not sure what is going on, I have used this very procedure many times with success, as well as other people in my lab. Setting up PCR straight from the colonies or the liquid cultures results in no bands, and PCR on the purified plasmid only results in large bands (plasmid). I don't think there is anything wrong with my PCR product, as cloning the TOPO control PCR is not working either. I would appreciate any thoughts/suggestions. Thanks!

I am confused about M13 PCR. can you clarify?

I'm assuming M13 PCR means you are using M13 primers that will only bind to the vector. Have you tried PCR with your primers specific for your insert? Or actually, any other set of primers, what about one M13 (or T7, or SP6), and one specific for your insert?
It's a long shot but it might be that your fragment does not have the required A overhang (despite being amplified with Taq) I usually amplify my gene of interest with Pfu and then treat with Taq + dATP for 15 min to make sure the overhang As are there. Works everytime for t-vectors.

If you are getting a good quantity of white colonies, then the problem is with the PCR. Add a little bit more of primers and template. For the pcr cycle add more time to denature and anneling step (remember that the strand must be open so the area is available to the enzyme and to the primers. Also can add PCR enhancers like DMSO, Tween 20, betain.

Sorry - yes, by M13 PCR I mean using the M13 primers specific to the vector. I did also try using primers specific to my DNA insert without any luck. Thank you for the suggestions about the PCR cycles, I will try modifying that today. I will also check about the PCR enhancers.

With vector based primers, you should get a (short) PCR band even without the insert. This means your PCR is not working, for one of many reasons. If you are a novice at PCR, then I'd recommend starting with a pre-made master mix. There are fewer things to go wrong. If you tell us how you are doing it (components of the reaction (exactly), amounts of primer, template, cycling conditions, we can likely identify the problem.

Thanks to everyone for the input - turns out there were a number of things that went wrong. Somehow we ended up with a bad batch of Ampicillin, so switching to Kanamycin plates helped dramatically. Also, my samples (which came from another lab) consisted of three sets. The set I began with and spent all of the time trouble-shooting was non-amplified, and not ready for cloning. Modifying my PCR protocol as suggested and using Kanamycin plates led to the controls working, as well as the other two sets. Thanks again!