View Single Post
Old 04-14-2019, 03:07 PM   #1
SDPA_Pet
Senior Member
 
Location: US

Join Date: Apr 2013
Posts: 222
Default Illumina MiSeq 16S sequencing EMP protocol questions

Hi, I'm going to do some 16S barcoded PCR following the EMP standard protocol here (http://www.earthmicrobiome.org/proto...standards/16s/).

The PCR potocol seems straight-forward. However, I am confused about what they say this on the website -- " For running these libraries on the MiSeq and HiSeq, please make sure you read the supplementary methods of Caporaso et al. (2012). You will need to make your sample more complex by adding 510% PhiX to your run."

I have a few questions:

1>I google the Caporaso paper and it seems PhiX is some kind of control?

2> At which step, should I add this PhiX? Do I have to add it or it is optional? Do I need to add this into my PCR master mix? -- add it in to every samples?

3>5-10% PhiX is the final concentration of PhiX or the volume of concentration the original PhiX?

4> Where can I order the PhiX or Is this a kit?
SDPA_Pet is offline   Reply With Quote