best gel concentration and voltage - (Aug/13/2005 )
hi all. I just want to ask your opinion if i am going to run a pcr product with target bands of 250, 350 and 400 bp for exmple ( from multiplex pcr), what agarose concentration, voltage and amp condition will I use, to get better bands.
How about if i just want to quantify my isolated dna in agarose gel., what voltage should i use and agarose concentration?
I am using 0.7% gel for my PCR products and running at 100V, 13ampere. Not so sure about quantification thingy though.
For a good separation I would go to a 3% gel or maybe higher and let it run for a long time? You can try it all yourself with several concentrations of gels and use a DNA marker for small fragment and use conditions that suit you best (meaning the time you have to let your gel run if you have a busy day and so on).
For myself I get quite good results by using 2,5-3% gels and let them run @ 100 Volts (about 15 cm of TAE-buffer and gel between + and - pole) for about an hour or so.
I agree with the above- I think about 2.5%-3.0% agarose should do it.
If diffusion (fuzzy bands) still presents a problem, you could reduce running time by cranking it up to ~0.25 Amp. But you should pre-chill the buffer and gel and run it in a coldroom to stop the gel melting!