Usually, I use a time of 120 minutes and 120 V, but I am testing and standardizing new primers and I realized that perhaps the sample might have run off the gel. I would like to know if you use any protocol considering base pairs to decide on the time and voltage.
There is no fixed time or voltage as conditions are set depending on the size of the band and the sizes of any other amplimers that need to be separated from the band of interest. The percentage of the agarose gel is important depending on the band sizes to be separated. I use 5v/cm where the length is the distance between the electrodes in the gel tand but this can be reduced when separating closely sized amplimers or increased if just checking for amplification. Small fragments are better run in cold gels to minimise thermal diffusion, A rough guide as to waht percentage gel should be used to run pcr products/restriction fragments is
%gel Range kb
0.3 5 - 60
0.5 1 – 20
0.7 0.8 – 10
0.9 0.5 – 7
1.2 0.4 – 6
1.5 0.2 – 3
2.0 0.1 – 2
- Badges
- Science topic