Cloning w/pCR4 TOPO vector - problems obtaining clones (Jun/21/2007 )

I am a biochemist only moonlighting as a molecular biologist.

An undergraduate student and I have been trying to clone a PCR generated site directed mutant of hen egg white lysozyme using the pCR4 TOPO vector sequencing kit from Invitrogen. The mutant contains a 5' end designed for the extracellualr expression of the protein using the yeast vector pPICZ alphaA also from Invitrogen. In addition, the 3' end has a silent mutation incorporating the Xba I restriction site. Finally, the site-directed mutant is N77H.

I have used the kit previously to generate a number of mutants of lysozyme and I have had little problems getting transformed bacteria (Top10 E. coli, also supplied by Invitrogen). We have not been successful transforming bacteria with the N77H mutant after mutliple attempts. We generate overlapping fragments about the site of mutation then use PCR to anneal the fragments and incorporate the 5' end for extracellular expression. Agarose gels indicate that we have a fragment of about 400 bp that correspsonds to the expected size of our gene.

We have used AmpliTaq gold. We have used GreenMaster Mix from Promega. We have added our PCR gene product directly after PCR. We have purified our PCR product. We have tried an excess of gene:pCR4 TOPO. We have used a 1:1 molar ratio of gene:PCR4 TOPO. We have purchased a new kit thinking the vector may have degraded with multiple melt, freeze cycles. I am at my wits end. I would really appreciate any suggestions.

Thanks

this is a long short, but can you check and see if your 400bp gene is in frame with the lacZ-ccdB gene. Sometimes the insert is inframe with the gene it was suppose to disrupt, which in this case results in the expression of a fusion-ccdB protein that proceeds to kill the cell.

There is not enough information here, to tell what when wrong. It is the exact details that the devil hides in. Need more information.
But have you tried increasing the ratio of insert to vector? 3:1 or even 5:1 ?

Nobody used proof reading enzymes did they? Only Taq produces A tailed products (only 70% are A tailed). Proof reading enzymes only have blunt end products

Still in situations where things don't work no matter how hard ones tries, then it is time to change strategy. You can add restriction sites to the end of your primers [Note restriction site require a few bp around it for it to be cleaved. (see NEB technical guide)]