I am currently trying to detect bacterial 16S rRNA DNA in human tissue to ultimately sequence the results. I conducted PCR on diluted samples (original concentration of DNA was between 100-500ng/ul) and when I ran it through gel electrophoresis I got a very faint band for 16S; however, when I attempted to sequence it, the concentration was too low.
I tried to then conduct PCR on the aforementioned PCR product without dilution to amplify the 16S target. I got a positive result (a clear, bright band) for my positive control (an E.coli sample); however, the other samples had a clear band at a shorter DNA template size (~200-400bp).
Is my 16S amplification being inhibited by the higher concentration of human DNA?
What will be the best way to isolate only the 16S DNA template in order to conduct PCR and get enough 16S PCR product to conduct sequencing?
Any advice will be greatly appreciated!!
you could purify your dna with akit designed for purifying small dna if you think the genomic dna is interfering but it is probably easier to design nested primers for your amplimer and run a nested pcr on the weak product ( heavily diluted and 10-20 cycles only) to get larger amounts of product
you could purify your dna with akit designed for purifying small dna if you think the genomic dna is interfering but it is probably easier to design nested primers for your amplimer and run a nested pcr on the weak product ( heavily diluted and 10-20 cycles only) to get larger amounts of product
If using a nested PCR you don't reach a large amounts of product, you can try to run on gel electrophoresis the product of the first PCR and then cut and purify from gel your band. So you should obtain a PCR product without interference of other genomic DNA that might inhibit the PCR. After the purification you can go on with the inner PCR.
Cartridge for rna purification can be used and nested pcr with 30 cycle and better to use real time .
Compared to conventional rt works better.
Regards
Aamir Javed
Instead of trying to separate the bacterial DNA from the human DNA, another approach would be to use the small amount of amplification from the diluted PCR for ligation. Clean up the reaction and concentrate the DNA, either using ethanol precipitation or a kit like the MinElute one from Qiagen. Blunt end ligate into a sequencing plasmid and use it to transform E. coli. You will probably need to combine several PCR reactions to get enough DNA to work with.
This will also help with another potential problem: 16S primers are usually universal primers, so if you have more than one species present in your samples (or you have one species with multiple, non-identical copies of the 16 gene), your PCR reaction may have multiple products. If you try this approach, keep all your transformants as they won't necessarily all be the same.
Regards,
Alison
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