I'm trying to do a library preparation for 16S rRNA microbiome profiling for different samples types using the V3-V4 primers' sequence recommended by Illumina. Some of my samples show no product at all including some of the stool samples while some from the same group show a perfect band on 1% agarose gel. I'm using NEB Q5 Hi-Fidelity DNA Polymerase which has been used in such work in previous studies and it shows a band on the gel in many samples and when I tried it with pure bacterial culture as a trial to confirm that it's working. I'm using the Purelink Microbiome Purification Kit for microbial DNA extraction. What should I do for the troubleshooting and how to obtain the best PCR product out of my samples?
Hi! Sometimes there are molecules that "survive" to the cleaning steps of the DNA extraction that can inhibit the PCR reaction. Try to dilute your DNAs to have ratios of 1:25, 1:50 or 1:100. Then, repeat the PCRs and good luck :)
Note: I assume that you measured your DNAs and that you know that you have enough DNA.
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