Ten sweet potato tuber samples from different locations were used for DNA extraction of endophytic bacteria .Small portion of frozen sweet potato tubers were cut and minced to a fine powder with mortar and pestle in liquid nitrogen. Then 0.1g of each sample was taken for extraction. The total chromosomal DNA was extracted by using ISOPLANT וו kit (Nippon Gene Co., Ltd, Japan) and used as a template.Then I tried for amplification of 16S rRNA for ten samples, all samples were amplified. But when I tried for Nifh gene amplification ,no sample was amplified. I used Pol F and Pol R primers and Prime Taq DNA polymerase .Please can I get any help for amplification of Nifh gene for my mentioned case?
Hi,
You probably amplified 16S rDNA which is present in several hundred copies in the genome. However, the sensitivity of your PCR could be insufficient for nifH. Have you checked the DNA quality? Is the concentration high enough? You could use another single copy gene as positive control in your PCR.
Are you sure that your primers bind to the DNA of all extracts? There could be sequence differences between your genotypes.
Best,
Boas
Dear Boas Pucker,
Thanks a lot for your suggestion.I will try that.
With best regards
Md. Arshad Ali
I think, those primers work well with 16S rRNA genes, but not with the nifH gene.
did you align primer sequences with existing sequences of nifH gene of sweet potato or similar plants? then, i think- you can have some ideas about successful amplification.
if not, then you have to design a new set of primers for PCR amplification.
Best of luck.
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