After doing PCR with SSR primers, should the product be analyzed on PAGE or Agarose and what are the electrophoresis conditions?
depends on the application, however the higher the resolution the better the results. on agarose you may get a one thick fragment which separates well on PAGE. that may be a defining quality especially if you would be looking into heterozygosity. I have used SSRs linked to important genes as tags for those genes provided they were closely linked.
Hi
It will depend particularly on the Band size of the PCR products. Generally SSR products need to be resolved on PAGE but also agarose gels could be used. Infect our group using Agarose and getting good resolution. In my opinion first give a try to Agarose if you find good resolution otherwise go for PAGE.
Use metaphor agarose gel from Lonza, USA. These gels will give fine resolution comparable to PAGE.
Hi
Sometimes in agarose gel 1.5-3% you can see polymorphisms . So first check it if you can not see resolution bands this is better you use metaphor or amresco agarose.
Hi!
Hope you might have come to some conclusion, my answer was little late.
It depends on how accurately you want see the difference. For eg. some times if you want to analyze even single base pair difference that you can not see on agarose gel. Then you should go for GeneScan (PE Biosystems) or MegaBACE 1000 (Amersham Biosciences).
Especially SSRs are smaller fragments so you have to use thicker gels 1.5 to 2% agarose gels, some times you clearly see variation if you use agarose gels some times they appear same but variation would be there. If you reduce the agarose fragments will run away. Using PAGE also good.
You mentioned about electrophoresis conditions this may vary from person to person, some body use TBE, some will use TAE. Some people adjust timer and forget about the gel the bands loose their sharpness, they diffuse. Gel picture does not look good. Though higher the % of agarose run little higher voltage and keep an eye on that.
Basing on your work exactly what you want to analyze choose the program and design the experiment.
The simplest way is to use metaphor agarose of 1.5 -2% for SSR analysis. If the amplicons are of smaller size then PAGE is better though a bit tedious in labs with limited facility
If the difference between SSR products are 10 base pairs or more, you can gel agarose 2-2.5% and for less 10 base pair you need PAGE.
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