Colony PCR - (Nov/27/2009 )

Dear all,

Recently, I am struggling with colony PCR to confirm the correct gene disruption in yeast. I have no problem when I did colony PCR to verify that my yeast strain has the URA3 gene. But when I do colony PCR to verify the correct deletion, I've never gotten any PCR products. I used the primers annealing to the promoter and terminator regions. I did colony PCR with these primers. No PCR products were obtained. I did a positive control with the primers annealing to the URA3 gene, I got nice products which means that the PCR reaction should have worked.

I am thinking that there is something wrong with the primers I designed. Could anybody tell me any tips about designing primers for yeast colony PCR, something like the primer length and Tm.

Thanks a lot for any suggestions in advance.

Pleazzzzzzzzzzzzzzzzzzzzzzzzzz. Could anybody help me?

yuer on Nov 28 2009, 05:44 AM said:

There is nothing special about colony PCR primers. The same rules apply as for regular PCR. Just make sure you are not using too low a Tm or you will get non-specific products. Did you check if your primers are having secondary structure issues or whether they are dimerizing or binding to each other? Any sequence analysis software can do this for you.
I would simply design a couple of new sets of primers at different positions and try again. Or, the other possibility is that the primer binding sites ( one or both) are missing in your yeast colonies. Mabe your deletion caused your primer binding regions to disappear too. Try using primers outside this region. If you can isolate yeast DNA, you could try restriction digestion using sites inside the deletion region to check if that portion is still around or not.

Dear Lotus,

Thank you so much for your kind reply.

The length of my primers are about 20bp. Tm for both of them is about 55 degrees (celsius). Both primers don't have secondary structures as I check with the IDT website. I did colony PCR with these primers in the wt strain which should have the gene, but no PCR products were found. I don't know what happened. The gene is about 1.5kb. These two primers should be annealed in the promoter or terminator region. So the total length is about 2.5kb. I don't know if it is because the length I amplified is too long.

I will design primers again as you suggested. Thanks again and hope get more advice.