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SB buffer and agarose: Fragments less than 100 bp

sb agarose small fragment buffer

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#1 Kikyou_

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Posted 07 November 2011 - 09:15 AM

Hi all.

I have a problem with a SNP, my genotyping bands are 70 and 40 bp. Try running in polyacrylamide, but I get a different pattern of restriction than expected. The person whopublishes recommended me a 2% agarose and buffer SB, but I have some doubts, bandsare going to see if they are so small? At what voltage I can run it? and for how long? Does the gel gets hot enough to run it cold? Bromide staining Does not give any problem?
Thanks for your answers

#2 allynspear

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Posted 07 November 2011 - 09:27 AM

Running in polyacrylamide should work fine, so if your banding is different than you expected, I would suspect a problem with the reaction first. A 20% TBE-PAGE should resolve those bands without a problem.

If you do want to try the agarose, I would recommend trying a NuSieve gel that is designed for higher resolution of small fragments, and follow their instructions for the fragment size you have.

If you just want to try 2% agarose with SB buffer, I would recommend starting at about 10-20 V/cm for your voltage, and just check your progress when the Xylene Cyanol dye is about 1/2 down the gel. Xylene Cyanol should run at about the 150 bp range, so your bands should be below it, but clear of the dye. Don't use a loading dye with Orange G in it, since Orange G will run at about 60-50 bp, and will be right on top of your bands. As for overheating, part of the advantage of SB buffers is that they supposedly give better resolution, faster running, and cooler running temperatures. You shouldn't have a problem with overheating if your buffers and gel are made up correctly. If you do notice a problem, it won't hurt to run the gel in a cold room, but again, this shouldn't be a problem.

Best of Luck.





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