3 replies to this topic

[wizzkid]

hi,
im having trouble introducing two mutations (2x F to A at positions 112/113) into my pcr product using the method overlap pcr. when i sent my results for sequencing they came back a 100% equal to the wild-type DNA sequence i used as template. I was very careful and did purify the intermediate PCR1a and PCR1b products before completeing the last PCR, PCR2.
Can anyone help me troubleshoot this?
would appreciate it  

[NemaToStella]

Hi Wizzkid,
what is it exactly that you're doing? Are you using TA cloning to clone the 'modified' PCR product or do you include restriction sites in the forward primer for PCR1a and the reverse primer of PCR1b? Do you have enough overlap in the primers for the mutation? Do you know whether you're transforming the template plasmid (assuming you use a plasmid template for the first PCR)? In that case a DpnI digest before transformation might help.

[Rsm]

Hi,

I second the usage of DpnI after first PCR. If you use gene-specific primer with RE sites attached, trace amounts of remaining plasmid will be sufficient to give you the wild-type gene after 2nd PCR. Or do you start from a PCR product as wild-type template? I usually don't purify my 1st PCR products. Just run a gel, mix 1a and 1b, add 1ul Dpn1, 37C for 1hr, take 1ul for 2nd PCR. After 2nd PCR you'd need to purify, especially if you want to use RE.

Best,

Minna

[wizzkid]

hi guys thanks for the replies, appreciate it.

No i didnt use TA cloning. For my DNA template i used HA-pcDNA3 containg my gene of interest. my primers PCR1a were A (forward)+C(reverse). A contained a restriction site and C contained my mutation. PCR1b primers were B(forward)+D(reverse). again D contained the restriction site and B the mutation. there should be a 23 bp overlap. After PCR2 i ran on a gel and was only looking for a bp band of approx 900 bp (HA-my gene) which i got. I then cut my pcr product and vector with the same RE, ligated them and transformed DH5alpha cells and grow up up colonies.

I like your suggestion of using DPN1