I'm working on optimizing three panels of sand lance for multiplex PCR. I'm essentially starting from scratch and first ran gradients on each individual marker to determine annealing temperature and size range, and I then created a multiplex plan.
The first panel went really well, and all the samples worked, which is why I can say that the DNA is fine.
For the second panel, I used the same samples, and am getting variable results. Some samples work pretty well, others have no amplification for any of the three markers within the panel. I'm not sure what my next step would be. This is my second try on this panel. The first was a standard PCR at the determined annealing temp, and the second time I tried a touchdown protocol and lowered the concentration of primers a bit.
Anyone have any suggestions on where to go from here?
DNA removes some MG ions from solution and this includes primers so I would try increasing the Mg concentration ( if using 1.5mM then try 2.5 or 3mM in the reaction)
The attached file may be of interest
Hey Kirby,
I have been through similar problems during my work with microsatellites. I agree with Paul, you can increase the Mg concentration. You can also try increasing the number of PCR cycles and the duration of final extension, it worked for me.
All the best.
Dear Kirby,
I think this is a really good paper for Multiplex PCR optimization.
Article Multiplex PCR: Critical Parameters and Step-by-Step Protocol
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