After restriction enzyme digestion of non purified PCR product with HhaI, many non specific bands observed along with specified product. PCR product size is 235bp
Ideally four specific bands should be observed (@ 89bp, 72bp, 67bp, 38bp, and some very smaller bands) But in this situtation bands starting from 235bp, then apprx. 200bp, 150bp, 100bp, etc are also observed along with speciifc bands, though intensity is less of non specific bands.
I am using 12% PAGE for studying SNPs.
Kindly help.
Hi Diji,
you say that the PCR product has not been purified. Are you certain that you are not suffering in the PCR from contamination of the desired product with (for instance), non-specific primer-dimer products?
An easy way to establish this would be to run an aliquot of your non-digested PCR product on the same gel as your digestion, this way you can see what the starting material is.
If it does transpire that you have both desired (235bp) and undesired products (200,150,100) from the PCR, can you optimise the PCR conditions to reduce the non-specific amplimers? Ie - change the annealing temperature, add supplements such as DMSO, change Mg2+ concentration, reduce the primer concentration or increase template concentration to change the reaction stoicheometry in favour of your desired product? Please feel free to give more details of what you have tried and we can try to discuss this here further. Best of luck, Robin
I have run my PCR product on 2% agarose gel but it does not show any non specific bands
Are you sure there isn't some incomplete digestion which could result in the additional bands? Maybe try incubating a little longer or adding some extra enzyme.
If you keep running into trouble, try gel extracting your PCR product and then digesting. There could be extra junk in there that you don't see (original template for example).
Hi there,
If your digest is not total, you might observe more bands than expected . I guess you know how cleavage sites are localized on the PCR fragment then you may anticipate the size of partial digest products. For instance the 100bp fragment could be the association of the 67 and 38 bp is they are neighbors in the PCR product and if the cleavage site hasn't been processed. BTW the addition of your cleavage products is more than the initial poduct...
So my suggestion is to digest for a longer time to have a complete digest profile.
OK. I will definitely consider your suggestions. Thanks for the support.
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