Hi all,
I am trying to purify from PAGE gels (with or without UREA) RNA/DNA fragments which lengths are between (for instance) 40 nt and 60 nt.
What I though was to produce some sort of home-made ladder that shows 2 bands for the limits I need and then cut between these two (in another well of course). Therefore I ordered 2 DNA primers, one of 40 and the other of 60 nt and I run them on a PAGe gel to see how they are migrating. What I got was only a smir (see figure).
Is this because my primer was perhaps too concentrated or I am just doing something silly? Do you have better suggestions on how to track specific lengths on a PAGe gel?
Thanks!
Lorenzo Gallicchio
In general, electrophoresis allows us to determine how many distinct DNA fragments are present in a sample and how big they are in relation to one another. We can also establish the absolute size of a piece of DNA by comparing it to a standard "yardstick" made out of known-size DNA fragments.
This tutorial might be useful, have a look: https://www.youtube.com/watch?v=eDmaBtxym30
If you are separating single stranded oligos of this size then a 6 or 7% denaturing (7Molar urea) PAGE will work well but oligos this long will form secondary structures and solvation effects will make native PAGE very messy. Attached is a protocol (old) from Harvard university you may find interesting. I assume that the length of dna that you are detecting is also single stranded or oligos will not be appropriate markers and for double stranded fragments it may be better to double digest a pcr amplimer or vector. Tracker dyes run at very small sizes which will also give you an idea where your bands are running.
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