I have to separate small cDNA fragments (160-180 bp) on agarose gel that will subsequently be used for sequencing. Any recommendation for which buffer is appropriate for running the gel- TAE or TBE? I am looking for a slower migration and better resolution of small-size DNA fragments.
Also, what would be the preferred voltage and time for gel run?
Dear Mansi Srivastava ,
According to Brody et al. 2004 Biotechniques 37: 598-60. What you really want is to run the gel as FAST as possible without the heating, and you just cannot do that with the usual TAE/TBE buffers. They are really terrible for electrophoresis, because they are too salty (thus the thermal heating) and the contain Tris, which makes the problem worse, because Tris breaks down during the run and makes the conductivity problem and heating worse in a positive feedback loop.
By simply switching buffers, you can run very fast gels (say, 1000V or 100 V/cm). As Brody et al show, very small fragments (24, 40, 60 80, 100 bp), normally requiring polyacrylamide, could be resolved in1 mM lithium boric acid (or 1 mM sodium boric acid) in a 3% agarose gel in about 5 minutes (100 V/cm). Make sure you use a good quality agarose with a low EEO value. You will also need to make sure you have reasonably low salt in your sample plus load buffer/dye. If your sample+loading dye is very salty, DNA migration will be retarded until the salts diffuse away.
Brody et al also have recommendations for the best simple salts and concentrations for separating other DNA size ranges (and RNA). But basically, either lithium (or sodium) boric acid in concentrations ranging from 1 mM to 10 mM will work for almost all your electrophoresis needs. You will save a lot of time on electrophoresis and on buffer preparation, as well as money, because the solutions are simple, low concentration, and cheap. You can buy the concentrated electrophoresis media and loading buffer pre-made.
TBE is the better choice ! TAE gave better resolution for large fragments and TBE works best for smaller fragment it also doesn't heat up as fast as the TAE.
As for the agarose concentration 2% would be fine we use this routinely for all the PCR analysis for classical gels run on a Mupid we use a voltage of 135 Volts and a 30mn run
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