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Overlap PCR - (Oct/09/2005 )

Dear all..

I'm currently struggling for my overlap PCR. I need to join up 3 exons. I managed to get exon A join with exon B. But i can't get my exon (A+cool.gif join with exon C. i tried gradient for Tm. I had tried Mg Optimization. I even change my polymerase.

Any suggestion from the floor? Thanks


Have you tried B+C and then adding A?

Are the outermost primers compatible? If you do a PCR with the sense primer for exon A and the antisense primer for exon C (assuming your gene is organised in that direction), do you get any results then?

Can't you also try RT-PCR of your entire gene?


yes.. I have overlapped exon (A+cool.gif, exon (B+C).
From the PCR products, i gel purified them and started another round of overlap. Just that when i tried to overlap (A+cool.gif + C or the A + (B+C), then nothing.

pls advise. thanks