I have been struggling to resolve a 4.8kb for a sub-cloning experiment from a 2.2kb band (other smaller fragments haven't been an issue).
When increasing percentage of low melting agarose gel (1.5% low melting or 1% low melting) the bands and ladder do not separate enough, when running on lower voltage such as 60-80V, for 2-3hours. Increasing voltage would melt the low-melting agarose.
But when using the normal agarose and increasing voltage (90-110V) there are smileys / smears which would affect the purity of the 4.8kb band I actually want.
Does anyone have any advice on what the best strategy is for a clearer/ crisper separation of bands?
Thanks!
run a 0.8% gel using cold buffer preferably in a cold room if one is available but run for much longer and at a lower voltage eg 40V for 4-6 hours. Time is less important than good separation here
Hi,
For gel purification you can get away with standard agarose (I prefer it actually, especially at a lower percentage). Paul is right about 0.8% gels and the lower voltage with a longer run. Since I'm guessing you are pouring your gels, I also would suggest using the same buffer for your gel as you are running in the gel box--i.e. fresh buffers all around from the same lot. We have evaporation challenges in Utah that sometimes alter the salt between the gel and buffer that may cause problems. This can cause frowns, smiles, and/or separation issues (depending who has been using the gel box, and whether they used water or buffer to make up the volumes).
Good luck,
Shale
Just like Shale and Paul said, all needed is to modify your method by reducing the low melting Agarose and increasing the electrophoresing time.
Agree with Paul Rutland and Shale Dames. It is recommended to use good quality agarose like SIGMA molecular biology grade to prepare 0.8% gel.
You can used normal agarose at 0.8% as some have suggested but use voltage 30 and lower your current from the one you were perviously using
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