Dear all,
I normally apply GTG5 fingerprinting to genotype a set of bacteria in our group collection. I was being successful from the beginning, but now the reactions do not work, just giving a smear or a low number of bands that is not consistent with the previous results.
I started also to observed a white mucous precipitate after PCR-reaction has finished.
Concerning the PCR mixture or PCR program, I did not change anything. I just observed that when gDNA is not freshly extracted, the reaction tend to fail, but if I just did the gDNA extraction, then I used to do not have any problem.
Please, have anyone an idea why the reaction is now not working? What can be that white precipitate?
Thank you in advance for your help
Do you resuspend/elute your final prep of gDNA in TE buffer or water? If in water the solution tends to become acidic which will degrade your DNA over time.
Hello Marcus,
Thanks for your answer. I do resuspend in TE buffer, so that can't be the problem.
I am wondering if the BSA I add might be making the trouble. It might be not in good condition might react with DNA and form that precipitate. What do you think?
Marcus: Change the BSA you are using. Take the fresh or of the different co.
BSA and DMSO are normally used to enhance the yield of the PCR and get more intense PCR products.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466135/
https://www.researchgate.net/post/What_exactly_is_the_function_of_DMSO_in_PCR
https://www.researchgate.net/post/What_is_the_function_of_BSA_in_PCR_products_of_no_amplification
Cheers
Perhaps if the addition of BSA proves a constant problem either 1) simply remove the BSA and optimise your PCR conditions with Mg2+ and temperature alone or 2) make up a BSA solution with sodium azide (0.1% w/v) before adding to the DNA. The azide should not affect your PCR.
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