Hi Ahsan Elahi
This is an unusual case, since usually people get less bp product, for which various reasons can be justified.
If you can give more details about your work and the experiments you perform in lab, we can have a clear cut thinking.
Some of the probable reasons:
- Primer cross-contamination
- Did you analyze the primer In-silico? Maybe that can say if your primer will give 700 or 530bp.
If any further assistance is required, feel free to message on RG!
Cheers!
Hi Ahsan,
It possible in some cases:
1- When primers are universal : e.g. primers for rDNA ITS-2 give different product sizes for different animal species.
2- It could be a mutation in the target.
3- The primers aren't enough specific... etc.
Any way to have a product of 700 is better than that of 530bp. If I were you, I will sequence this product of 700bp using the same primers to see what is it.
hi Ahsan,
If you are targeting mRNA, it could be that there are issues of genomic DNA contamination. If you have genomic DNA contamination, you would expect both the 500bp and 700bp to be present but if the template concentration is too high, one product might be prominent.
@Aravind Kumar N, I have self designed the primers...primers are universel for the Cryptosporidium Specie.. I have blast the primers on Ncbi and it shows product length range from 524 to 539 with different species.
P. S Primers are insilico validated and have no hairpin formation issue...
Ahsan Elahi
Hello!
If the primer details can be shared, I can perhaps validate the primers and may be able to troubleshoot.
If possible, kindly message me on RG!
Cheers!
first repeat
if observe the same band you would better to change yor primer
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