Microsatellite PCR Problem

[TKTK]

Hi everyone,

I'm working with Douglas fir microsatellites - I have six loci that I optimized previously for primer conc, MgCl2, temp and cycle number. I amplified about 300 samples without trouble, then took a month or so to do some genotyping. Since I've started trying to run the reaction again, I have been getting a ton of background amplification sometimes with my primers, and now recently no amplification. I have ordered new primers, and replaced buffers and dNTP's (though I haven't ordered NEW dNTP's). I've also checked these primers against DNA we've previously amplified and my current problem is showing up. Any suggestions?

[dzanches]

Originally Posted by TKTK

Hi everyone,

I'm working with Douglas fir microsatellites - I have six loci that I optimized previously for primer conc, MgCl2, temp and cycle number. I amplified about 300 samples without trouble, then took a month or so to do some genotyping. Since I've started trying to run the reaction again, I have been getting a ton of background amplification sometimes with my primers, and now recently no amplification. I have ordered new primers, and replaced buffers and dNTP's (though I haven't ordered NEW dNTP's). I've also checked these primers against DNA we've previously amplified and my current problem is showing up. Any suggestions?

Well, it seems you have problems with unspecific amplification. I also had this problem in my lab: first everything went OK, then it suddenly did not work. We solved this problem by buying new primers, from a different company, and got PCR kits also from another company. Somehow we solved the issue, but there were still some unspecific bands we did not see in the first runs near the expected amplicons. We then changed the standard PCR protocol for a touchdown one and made a new DNA extraction by the HOTSHOT method instead of the chelex approach (I work with insects and fortunately have more than enough tissue to do several extractions). Since then I get the usual unspecific amplifications below 50bp in some primers but always clear bands where they are expected to be, like in your good gel figure. And interestingly some of them are not the same size than in the first runs.

Hope this helps,

daniel