Does using High fidelity polymerase helps in the amplification of 16S rRNA gene from the total community DNA (sediment, water) if the normal Taq polymerase don't work ?
Hi dear Ashish
You can read the source in below which you can of problem be resolved
Article Detection of 16S r RNA gene of Helicobacter pylori in patien...
https://www.researchgate.net/publication/304783575_Molecular_Detection_of_IS6110_of_Mycobacterium_tuberculosis_in_pulmonary_tuberculosis_and_tuberculous_pleuritisEnter_title
Dear Ashish
It sounds as though you are having issues with the specificity of your PCR set up. Though I would need to know more, what are you seeing when you run your results in a gel?
Some of the main factors affecting specificity that you ought to consider are:
1. The quality of the primers being used and primer concentration
2. Annealing temeperature
3. The type of polymerase you are using, probably best to use a hot-start
polymerase rather than a standard polymerase. High-fidelity polymerases work to ensure that the sequence is amplified correctly (reduces misincorporation rate) but do not necessarily confer higher specificity.
4. The concentration of polymerase
5. the ratio of dNTP: Mg2+
These are just a few parameters to consider , without an idea of what results you are getting it's difficult to suggest what exactly the problem might be.
I hope this helps with regards to troubleshooting your issue.
Kind Regards
Benjamin
It sounds like your PCR reaction is not working. I would recommend diluting your enviromental DNA to 1:10 before running PCR. It works in many samples, as it also dilutes the PCR inhibitors. Second thing your could also try adding BSA in our reaction. There is no exact concentration of BSA, you need play around and check with different concentration that fits your sample. I hope this will solve your issue. I would not recommend using high fidelity polymerase.
Regards
Rumakanta
Hi dear Ashish
You can read the source in below which you can of problem be resolved
Article Detection of 16S r RNA gene of Helicobacter pylori in patien...
https://www.researchgate.net/publication/304783575_Molecular_Detection_of_IS6110_of_Mycobacterium_tuberculosis_in_pulmonary_tuberculosis_and_tuberculous_pleuritisEnter_title
I am agree with Rumakanta try diluting your enviromental DNA to 1:10 before running PCR
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