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
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%)
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.
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
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.
once i used 3% agarose gel and run it for 3 hours with 50 volt...it worked for me ..:)
Lower temperature also improves resolution. You could circulate your buffer through tubing in an ice bath.
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 on Mon Apr 14 18:56:17 2014 said:
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...
just curious: if it is cheaper and better, than why do people keep using other buffers than the lithium borate?
lyok on Sat Apr 26 18:58:20 2014 said:
hobglobin on Mon Apr 14 18:56:17 2014 said:
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...
just curious: if it is cheaper and better, than why do people keep using other buffers than the lithium borate?
I guess mostly out of tradition or habit, they learnt gel electrophoresis with TAE or TBE, have the buffers and recipes and protocols and don't need to think about with standard work. A few drawbacks are there with such borate buffers too: high molecular DNA is not resolved as well, some dyes are not working. Perhaps also issues with a few downstream applications, which I don't know (DNA extraction and purification out of the gel not).
And finally it's a not that well known and common buffer.