16S PCR Cleanup: MoBio or Qiagen

[rsirois]

Hello all,

I will be generating 16S amplicons for use on the MiSeq platform. The article that I am basing my protocol on notes that they used the MoBio PCR clean-up kit to remove residual PCR "junk" from their amplicon products prior to sequencing. The kit is rather expensive for my lab and I know that we already have the Qiagen PCR Cleanup kit. Does anyone have any recommendations on which kit to use? How different are the kits from one another? Does one work better than the other? Will I get similar results?

Thanks,

Rachael

[ScottC]

Firstly, I'll say that I haven't used the MoBio kit. But, if you're just doing a regular PCR cleanup, I think nearly any PCR cleanup kit will be OK. We use either Qiagen or AmpureXP.

[jertlse]

Hi rsirois
In my lab we use Ampure Xp for all the clean up and purification.
I have to generate 16S amplicons for MiSeq too, can you give me the reference of the article that you talk about?

Thanks

Jer

[rsirois]

Hi Jer,

Thank you for your reply. As far as the reference goes - I have been basing my PCR protocol off of that described in Caporaso et al., 2012 (Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms). It provides a very thorough supplementary methods and I know many people in this forum have used this paper. Best of luck.

-Rachael

[kkrishnan]

Hi, I was wondering if after the PCR step, we need to perform a size selection on the gel? My PCR product is not just one band (even with the V4 primers from Caporaso). If only a column cleanup is done (as performed in previous studies followed by pooling then size selection just before sequencing) and libraries pooled then isn't that inaccurate? However gel extraction of several libraries seems not-so-high throughput...Any suggestions/advice is greatly appreciated. Thanks!