August 13, 2011 at 1:00 am #15270EtrynParticipant
I’m pretty new to performing gel electrophoresis and I’ve been having some issues. Many people have recently left the staff at my lab, so we’re a little short on expertise in this area. My main issue is that I’m getting poor separation for the bands in my low molecular weight ladder.
I’m following the same protocol used by former members of my lab, and I’ve seen pictures of their gels and the ladders look perfect. The 2 or 3 largest bands in my ladder are distinct, but all the small bands are fuzzy and indistinguishable. Everyone running gels in my lab right now is having this problem.
We are using a New England Biolabs Low Molecular Weight Ladder (which is, as far as I know, the same ladder we’ve always used in my lab) and a 3.5% agarose gel made with TAE buffer. I’ve been running my gels at 60 volts. NEB recommends loading .5 ug of ladder into the well along with loading dye, which is what we’ve been doing.
I’m intending on experimenting with different voltages/agarose concentrations, and maybe ordering fresh ladder and seeing if that is the problem. I thought I’d also ask these forums: is there something we’re all overlooking that might be causing bad ladder separation?
Thanks so much!
August 13, 2011 at 2:30 pm #105917canalonParticipant
3.5% Agarose? IS there a reason to use such high concentration? I usually work between 0.8% and 2%. And If I remember correctly getting a nice and evenly melted gel at 2% is not easy.
But whatever concentration you are working on:
– check that the heating do not cause too much evaporation (make a mark on your flask before melting and add milliQ H20 back to the level if necessary
– Check that the solution is perfectly clear, you have to be quite careful, but by swirling it in front of a light source (or window) you can check for the absence of granule and filaments that are transparent, but with a slightly different refraction index. Those are unmelted agarose granules that do not have the same concentration than the rest of the gel. the presence of such granules in your gel is going to ruin the migration pattern (DNA is going to slow down in spots.
Hope this helps
November 6, 2011 at 12:19 pm #107730JackBeanParticipant
in our lab is used even 4% agarose gel for separation of fragments like 20-200 bp after real-time PCR. What I remember (I’m not working with this), the gel is not much clear, but stays kind of yeallowish. But for sure check for the clumps as canalon suggested.
What range of sizes are you trying to separate? Maybe try to post some picture of your gel, so we can see the problem 😉
November 6, 2011 at 10:03 pm #107742
November 8, 2011 at 1:55 am #107803canalonParticipantquote Cat:
Depends on the application, but using Low melting agarose for regular electrophoresis is a waste of money. The OP has not provided us with any more information about what (s)he is doing, but unless they are looking for tiny difference in short fragment, I cannot see why they are using such a high agarose concentration. And if the differences are that small, it would make sense to run a polyacrylamide gel any way.
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