Sometimes I see the shadow like bands and its not true band. I want to know that what's the reason for it. I am using 2% gel for running genotyping samples
I have uploaded the gel picture in both background i.e black and white
You could have a polymorphic amplimer (CA or triplet typerepeat sequane) on one chromosome or the primer could be annealing in 2 places but you say this is not a real result. If you do gel electrophoresis on the same samples again do they give the same double band result?. If the primer is annealing in 2 places you could try raising the annealing temperature or adding dmso 5% final concentration to the pcr
Paul Rutland and Sa Chen Thank you for the great insight. Some time I got one band with same primers but different samples of mice. If there was issue of primer annealing in 2 places then the marker should have one band but unfortunately I also saw shadow bands of markers also. So I think I am making some mistake in gel concentration or running etc
When I say the primer is annealing in 2 places I had in mind the following. If the amplimer is 500 bases then the forward primer might be 1-20 and the reverse 500-480 but if the reverse primer can also anneal less well at 570-550 then you might get 2 bands on all amplimers. Increasing the annealing temperature may minimise the second band. It may be that your annealing temperature is just on the level that sometimes gives 2 bands and sometimes only 1. This really does not look like a gel running problem to me. Good luck
Paul
Have you tried running a gradient of annealing temperatures? That can help identify the ideal temperature for your primers.
What size band are you expecting? Have you tried cutting out & sequencing the unexpected "mystery band"? That can tell you exactly what you have amplified.
Good luck!
Katie A S Burnette I have optimized before but was not so ideal sometimes I got single bands and sometime double band.
I am expecting band of 1295bp band , after gel purification i usually send for sequencing but not this one.
Thank you so much.
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