Actually I am having issue with double purified PCR bands for gel electrophoresis..
You cannot send the mixed pcr product for sequencing. It will look like a mess of peaks. Your main options are
1 cut the bansd out of the gel and column purify each band separately and sequence. This is best if you think that the 2 bands are related like normal and deleted template amplimers
or 2 add dmso up to about 8% maximum and hope that the increased stringency removes the wrong band
or 3 increase the annealing temperature until the wrong band disappears,
2 and 3 deal with the problem of the 2 bands not being rellated and one band just being caused by inappropriate primer binding.
Do not need to worry about this. It is due to nonspecific binding and use of extra taq polymerase. Again, run gel ELECTROPHORESIS aboutT 15 minutes,it will disappear.
What size product were you expecting? Do you get similar results with a different set of primers for the same locus?
If you are working with a gene family or polyploid organism, it's hard to design primers that are specific to just one locus.
The only way to know exactly what you have is to cut & sequence the bands separately.
Good luck!
The appearance of double bands is due to nonspecific binding. You can play around with annealing temperature according to your primer.
Hi I ran a gel today, and my DNA ladder looked similar to yours, except it had a slight tail. I was wondering if you knew the reason why this happens as I'm not sure.
- Badges
- Science topic