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TOPO TA cloning - (Feb/02/2006 )

I am trying to insert a 413 bp PCR product into PCR2.1 TOPO. I have used the GenElute Cleanup kit on a fresh PCR product and. After growing on ampicillin selective plates and mini-preps, the restriction digest (using EcoR I) gel shows plasmid but no insert. Someone else in the lab is using the pCR4 TOPO on a different PCR product and is getting exactly the same result. We know the ampicillin is working as is the restriction enzyme. We have attempted this on numerous occasions to no avail. Does anybody have any suggestions as to why the inserts are not ligating into the plasmids?
Thank you.



you could try to gel purify your product as an alternative. I usually do it this way with TOPO 2.1.
Another thing may be your PCR protocol:
Be sure to include a final step for 10-15 min. 72°C and Taq enzyme to get A' overhangs.
If you do your PCR with PFU you can also add Taq afterwards and extend your PCR product with A' overhangs
Good luck.


maybe should you have a look at this topic too