Hi Santoze,

the labelling of one of the primers from a couple won't change anything compared to "normal" PCR primers designing. designing, using and amplification are strictly same. storage and manipulation must limit light as possible, that's all since dyes are sensible. The only change will be the detection, probably on an automatic sequencer as ABI know to do.

fred

You can optimize your reactions as regular PCR but try to use minumum amount of dNTPs and primers to get better peak reads.

And you can save money by using less primer since the labelled ones usually cost more.

Thank you all.

Good luck and best wishes for your project.

dear Santoze, primers design for single primers will be the same as for regular PCR. the only difference is that when you order the primers, generally the forward is labelled with a fluorophore.

However, one of the advantages of microsatellites is multiplexing. it is worth optimizing multiplexes of up to 12 primer pairs that can be genotype in a single run. i fyou go down this path, I suggest you use quiagen multiplex PCR kit. the optimization of multiple primer pairs amplification requires that different PCR band sizes and fluorophores are used so that you are able to read multiple regions on the ABI. I hope this helps

Like others have stated, it shouldn't be too disruptive to your "normal PCR" protocol other than 1) you likely wont need to use as much primer in your mixes, and 2) you'll need to be sensitive with how you store and add your dye-labeled primers due to the influence of UV (degradation). One extra note that hasn't been suggested yet is that some fluorescent dyes amplify better than others (e.g. blue), meaning you might need to alter the amount of each dye-labeled primer you have depending on its specific dye. This however, is only a huge problem when multiplexing and will likely just require iteratively checking peaks after ABI analysis and adjusting as required. Good luck.

I quite agree with all the answers and suggestion. Good luck!