TOPO cloning problem - (Apr/12/2007 )

Dear colleagues,
I've run into a problem with a TOPO cloning reaction. I amplified my gene of interest using pfu with a forward primer that has CACC followed by the start codon. The PCR amplicon looks great, with appropriate band at 2.9kb. I cloned a diluted amplicon into the Invitrogen TOPO vector & got >100 colonies on a kanamycin plate. After mini-prepping 5 colonies & digesting with ASCI and running on a gel, I get a band at about 4.0kb, not 5.5-5.6kb in all 5 reactions. Have any of you run into this before? The only thing I can think of that happened is that an additional TOPO recognition site exists in the gene and 1.5kb are spliced out... Any suggestions?
Thanks!

"I amplified my gene of interest using pfu"
There's your problem. pfu gives blunt-ended PCR products, not the A overhangs needed for TA cloning. Try Roche Expand, it's a mix of pfu and Taq, so you'll get good fidelity and sticky ends. The 4kb bands you are getting are empty plasmid, which you should not be getting if your vector is in good condition, is it old or has it been thawed and frozen lots of times?

You can also treat your pfu products with a regular Taq. Just incubate you PCR product with 2mM of dATP, 1U Taq, 1xBuffer and your usual Mg concentration for 20min at 72ºC. After that you just need to clone it in your TOPO vector. We do that here in the lab and it works quite well. Sometimes, in you proof-reading polymerase manual there is even a protocol for doing this kind of treatment. Do check it.

Maybe it's directional topo cloning or blunt end topo cloning stead of TOPO-TA cloning?

ah, OK. But either way, a band at ~4kb means recircularised plasmid...

Have you gel purified your pcr-product, or purified it to get rid of primers? Maybe there's your problem, one of your primers contains the CACC that will anneal to the 4 base overhang in the vector leading to circularisation maybe?

I spent a long time messing with TOPO crap. I cannot recomend against it strongly enough. TOPO will not just snag the CACC overhang on the 5` end, but it may also be getting one internal in your gene. I left TOPO and went old school, that's why they call it old school, it works.

I guess it all depends on the gene of interest.
I spent a few months assembling a gene from different cDNA sources and for some of them the TOPO directional strategy would simply not work and thus I used the TOPO-TA vectors.. trial and error