That looks like a good set of results. A no template control would be good to check for contamination in the future and possibly run fewer cycles to get a cleaner result and slightly less product as very strong amplification product can spill over into the next well and look like an amplified but weak band so why do you need another test?

@ paul, thanks. So should i count sample 4,7,11 in lane 1 and sample 3 in lane 2 as heterozygous?

Md. Habibul Hasan Mazumder Hi

Yes these samples are difficult because the lower band is weak compared to the normal band. How can this happen?

Some possibilities are

1 Contamination of a reagent with previous pcr product. 45 cycles is a huge number of cycles and even the slightest contamination will amplify. This would often show in the no template control giving a positive amplification. Solution ..run another amplification with 30-35 cycles and include 2 NTC samples.

2 Gel loading problems where material from the samples either side have spilt over into the queried samples…the wells split a little on removing the comb or too large a sample was loaded. The last sample on the top has a weak upper band which might support this possibility.

3 The forward primer may sit over a polymorphism near its 3’ end. In this case some samples will amplify badly off one chromosome and give a weak amplimer so in this case these samples could be real heterozygotes. I suppose that a pcr from outside the deletion with a reverse primer inside the deletion would show either nothing ( deletion) or a short product ( normal) which might help.

4 In these samples the organism is chimaeric so cells exist either deleted or normal and your pcr reflects the mixture accurately

I would prefer to read these sample on a gel with NTC samples and ideally a space between each sample on a pcr with 35 not 45 cycles