I'm trying to determine parentage based upon microsatellite markers, and using agarose gel electrophoresis to visualise the different lengths. Fragment size is c.100bp and having trouble getting good enough resolution to see the different bands. Have been using 2.5-3% gel for 4hrs.
I would use acrylamide gels if it is a dinucleotide or trinucleotide repeat but that is not what you asked . Trying with agarose I would use a 2:1 mixture of low gelling agarose and ordinary grade agarose at 3% concentration. Cool the gel and the buffer in a fridge and run at half your normal running voltage ( although if you are running it for 4 hours you are probably already doing this). This cuts down heating of the gel and buffer and minimises thermal diffusion which broadens the bands of amplimer,If you have a cold 4c room then run the gel in the cold room. Check the gel early as band broadening will be minimal and running a shorter distance may be easier to read with clearer bands. If this does not work think again about acrylamide with silver staining. I know you can separate 100 and 97 bases on a 7cm acrylamide gel.#If you do run gels in a cold room when you take the power supply back out of the cold room leave it for 4 hours to warm up before using it as condensation on the pcbs can be a problem
Hello Abigail.
I would suggest you run polyacrylamid gels as Paul already did. Another idea is to run your fragments on a sequencer with labeled size markers in the same lane. That would give you the best resolution.
The fragment size is complicated since it's on the edge of what you can see with agarose, and over 2% you start losing resolution. I would use polyacrylamide. Look for the proper concentration of the gel and if/how much bisacrylamide to use. If you want to denature, use Urea. I'm sure there are better guidelines, but this is one I found.
https://www.alfa.com/media/PF_PolyacryamideGelsElectrophoresis_2016_V1-digi.pdf
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