I amplified a beta-tubulin gene fragment known to be polymorphic and used a published primer. though the author used Restriction enzymes to digest the pcr products. I cleaned the pcr products and after amplification, I find out that repeatedly there were consistently 2 bands at 100bp and 200bp on some sample, while other do not have against the expected 499bp on the agarose gel. please I need explanation for this.
The primers are not specialized.
DNA is not pure.
Defective primer design.
Incorrect program for temperature annealing.
Hi @Danamil Elisha Akafyi
Its looks like there might be some contamination in some reagent which differ in different samples.
The band intensity of the second band doesn't increase with the first band which might be because of some contamination.
Is the sample is laboratory generated?
As field samples might contain other oraganism and the primer is amplifying the other organism beta actin due to similarity in seq.
Hope u find it helpful
I think you should check again the primers first at PrimerBlast NCBI for make sure that is for your terget gene
From the picture you posted which I assume is from the paper you got the primers from it would appear that the bands at ~100bp and ~200bp are due to non-specific or off target amplification. Frequently published papers do not contain the optimised amplification parameters, but rather the initial ones before any optimisation. You can try to alter your Tm (do a gradient PCR) to see if you get the ~500bp band to appear and then find the optimal Tm.
I'm not sure what the purpose of the restriction digest was. Was this before or after the PCR amplification? If it was afterwards, it was likely to cheaply look for a SNP in the amplified band.
Anyway, maybe try to find some other primers since PCRs with that many off target bands make for very difficult downstream uses.
Your primers might be contain repeated sequences pattern along your gene.
You have to do blasting as they suggest, design your primer carefully.
Use only 1- 10 ng as the final concentration of your primers and increase annealing temperature.
Good luck
You mentioned repeatedly. Do you mean repeated the pcr?When you repeat the pcr, did you used template from purified pcr product or from original samples? Because it will differ sometimes. It is clear in the attached picture that you have dimers. Dimers can be avoided by decreasing the volume of primer. If you decided not to pcr again, purifying the pcr product by manipulating the reagents can do (to attach only the
targeted bp in the silica gel).
If the problem comes from the primer annealing step, then I will go with a gradient PCR to find the best temperature for annealing. But if it comes from contaminated template or primer solution, then I will start again fresh from a new working solution.
hope it helps
There possible areas to check
1: Purity of your sample
2: Primers designing
3: Primary and secondary stock preparation of your primers
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