Two-Step PCR for amplicon sequencing issue

[ashah]

Has anyone come up with a PCR mix for the final PCR that adds the Nextera indices and P7/P5 to the product?We bought the 96indices Nextera kit and are able to use them with variable efficiency for indexing our PCR product.

We are doing a two step PCR to sequence amplicons on the miseq. So first, we amplify with:
5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[Forward locus specific sequence]
and
5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[Reverse locus specific sequence]

Then, after cleanup, we tried using NEB Phusion HF along with the Nextera Index primers to add the indices, but are having variable efficiency/extra bands.

We originally had some leftover PCR mix (and PPC) from a Nextera Enzyme kit, but that ran out. Anybody already optimize the PCR conditions for a different taq? Also, what is PPC (Primer Cocktail)?

Any hints would be appreciated,
--AS

[ZWB]

We pretty much use the exact same protocol as you. We found that the best enzyme mix to use is FailSafe with Buffer E (EpiCentre). I've heard rumors that the buffer in the Nextera PCR mix is in fact FailSafe Buffer E anyway. For whatever reason Phusion doesn't work in our hands either.

Zach

[microgirl123]

I've been using the Phusion HF. I haven't done it enough yet to determine how variable it is. One problem I run into (we're a core facility and only perform the second step of PCR) is people submitting DNA from the first step quantified only on the Nanodrop. This means that I'm not always adding 50 ng of DNA to the second PCR.

I always see a second, slightly smaller, peak on the BioAnalyzer. I've been attributing this to some amplicons only having one full adapter added - it's only 5 cycles of PCR so it stands to reason that some of the amplicons only got one adapter added.

[Neiltje]

I am also trying this protocol but I'm running into problems.
My DNA is bisulfite treated (all non-methylated C's are converted to U) so I'm using an enzyme from KAPA that is especially designed for this type of DNA. My PCR works just fine when I'm using primers without the 5' overhang adaptor sequence. But when I use primers that do, all I get is primer dimers and no good PCR product. Does anyone have an idea how to fix this? I tried it on genomic DNA and on PCR DNA but neither works

[nucacidhunter]

Please see this thread: http://seqanswers.com/forums/showthread.php?t=43376