I am getting ok bands after first step PCR - measuring 1-2ng/ul, is this enough?
It should be enough. Normally we use as template 1 ul of the 1st PCR (15 cycles). The concentration of our DNA should be around 1-5ng/ul. We run the 2nd PCR for another 20 cyles. Please keep in mind that the total amount of cyles should be less than 40 in order to avoid amplification of bias...
Cheers!
There is no specific amount of DNA normally with Nextera library protocol you only need 5ul of your amplicon PCR for the index PCR. So there is no need to quantify at this point.
It could be enough. There are different library protocols and reagents; some are good for nanogram-level concentrations, but there are also library prep kits that can work for picogram concentrations. I believe that the quality and purity of the RNA extractions is more important.
Thank you everyone for your help with this. My starting DNA conc's, as quantified with Qubit are approx 50-300ng/ul, however a lot of this will obviously be host dsDNA, so i'm trying to understand how much microbial DNA after first step PCR amplification might be optimal? My samples range from 0.2ng/ul at the lowest, to 9ng/ul at the highest after this step, representing conc of V4 amplicons, as measured with Agilent Bioanalyzer, could this be enough?
How are you planning to make your libraries? The input concentration for prep methods vary. For Nextera XT it is 5ul of 0.2 ng/ul (1ng in total). Are you planning to purify the V4 amplicons?
Following up the question mentioned here... I believe it is better to ask in here instead of writing in a new page. After 1st PCR, I have a clear band and then, I performed gel extraction to purify that amplicon. I used 1ul from this purified 1st PCR product for the second PRC which is a barcoding for MiSeq but I got a smeary band. My band should be around 600bp which I have in some lanes as you can see in the attached file. The very right one is negative control in which you can see only primer dimers. However, in the samples lanes I have bands around 300bp too which I couldn't figure out what is the problem. In theory I performed second PCR from purified product of 1st PCR. Can any of you give me any suggestion for the second round of PCR? I did 40 cycles in total. Could that be a problem? Shall I decrease the number of cycles or shall I decrease the amount of DNA added to the second PCR? Thank you very much.
Perhaps an EXO-SAP purification step, after your gel purification, to make sure you are amplifying only ds amplicons from first PCR step.
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