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Altering gel voltage and run time. - (Feb/07/2014 )

Hi,

 

This is probably a pretty simple issue. I'm currently separating bands between 200 to 300 bp in length. At present I'm using a 2% gel set at 65V for 40 mins, however, the resolution isn't great. I understand that better resolution can come from decreasing the voltage and increasing the run time, but I can't seem to work out what the relationship between the two is. For instance, if I decreased the voltage to 60 what should the increase in the time be? 50 minutes? 60 minutes?

 

Many thanks in advance

-Jen_S-

In this situation better resolution can be achieved by increase the agarose gel concentration to 2.5% or even 3%(if you are careful and get the agaorse powder to disperse before melting it)

 

Other alternatives would be to use an acrylamide gel. (~10%)

-perneseblue-

I would also say to run it for as long as possible - without running them off the gel.  If you can, use Orange G loading dye, which runs at about 50-100 bp, rather than using bromophenol blue, so that you don't accidentally run off the gel.

-bob1-

Lower voltage will make the separation better, as for the time, I don't think there is accurate formula to calculate this, it is more based on past experience. I would just suggest both try different time points and see how they are

-lilin_hust-

you can get a volt-hour integrator (or a power supply that contains one) and match volt-hours for a good separation (as long as the gels are identical). it is (or was) for ief gels to ensure reproducibility.

-mdfenko-

once i used 3% agarose gel and run it for 3 hours with 50 volt...it worked for me ..:)

-MacGyver-

Lower temperature also improves resolution. You could circulate your buffer through tubing in an ice bath.

-phage434-

Not to forget a different buffer...e.g. Lithium borate, then you can increase voltage (150 V for a 2.5% gel should work) without heating up. This reduces running time (more convenient and less diffusion) and you get sharper bands.

And it's cheaper...

-hobglobin-