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

I tried amplifying the environmental DNA by using normal taq and high fidelity taq polymerase and got the amplification only in the reaction setup by using high fidelity polymerase. I think the high fidelity polymerase does work when the concentration of your environmental DNA is less i,e., 1-10ng/µl. The following rex. was setup:

5X Go Taq buffer-10µl

10mM dNTP- 1µl

25mM MgCl2- 6µl

63F (50pm) - 0.5µl

1387R (50pm) - 0.5µl

Taq (5U/µl) - 0.25µl

Template - amount shown in attached file

Nuclease free H2O - make up volume to 50µl