16S V4 amplicon concentration after first PCR step

[Egansbay]

Dear all

I am analyzing gut samples from fish using illumina's 16S protocol, amplifying the v4 hypervariable region. I am getting ok bands after the first PCR step, and a nice peak on the agilent bioanalyzer (approx. 350bp). I am wondering however, after purification of products, what is an acceptable concentration for each sample? I am getting concentrations of individual products of between 0.5 and 9ng/ul, with the average being around 2-4ng/ul per sample. Is this an acceptable concentration to proceed with?

Thank you very much in advance.

Kind regards

Egansbay

[microgirl123]

Hi Egansbay,

I usually quantify my first round PCR products with Qubit and then try to add 50 ng of total DNA to the second round of PCR for 5 cycles. If my concentrations are much too low to do that, I add up to 3 more cycles of PCR.

[Egansbay]

Thank you microgirl. So in your experience are my concentrations of product too low to proceed with second round pcr?

[microgirl123]

If you had good bands in the first place, I would go ahead and give it a try - you can always do a few samples first. I use an adaptation of the Illumina method with a 50 ul PCR volume, up to 15 ul of cleaned template, and 5-8 cycles of PCR.