I am determining my protocol to run a denaturing DNA PAGE gel to look at DNA fragments just under 100bp. I was wondering whether people commonly used a stacking gel to enable a good transition of products into the gel, and if so, what the constituents/concentration was of the stacking gel.
Nope. Never used a stacking gel for that. I've run DGGE, TGGE, and regular PAGE with DNA and always had one layer. The stacking layer (different pH) is really for proteins. If you have a copy of Molecular Cloning (what we used to call Maniantis) in the lab, there is a really nice piece in there about what a stacking layer does and how it helps with resolution of proteins. Depending on how many bands you want to resolve from one another, a regular 12-15% PAGE should do the trick.
Hi James, Thank you for your answer. I found that regular PAGE with a 12% gel and no stacking buffer did enable me to visualise the bands that I was interested in but overall sample migration and resolution of the smaller fragments (below 100bp) were better with a denaturing gel. I also ran the same samples on two Denaturing DNA PAGE gels- one with a stacking gel and one without. Even though the migration of the bands was exactly the same on both gels, the resolution and sharpness of the bands was much better when a stacking gel was added so I have stuck with that and got consistently good gels.
Good, glad you found something that works for you. At the end of the day, the most important thing is that what ever you do actually works.
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