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Old 03-08-2017, 08:36 AM   #1
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Location: Edinburgh

Join Date: Mar 2017
Posts: 4
Default 16s v4 region library preparation with negative control

I am about to run 16s v4 region for bacterial population comparison between groups.
I used Illumina nextera index and kit earlier.
But now I would like to save cost, so I will turn to custom primers and custom preparation.
So, the pipeline of labwork is.
1. PCR the 16s v4 region with primer from this reference.
2. Check the band in gel electrophoresis.
3. Clean the PCR product with Ampure xp beads.
4. Measuring concentration and size with Qubit broad range and agilent tapestation.
5. Normalise all samples to 4nM concentration and pool them together into one tube.

Is this the correct workflow? or Should I change or add something else?

then my bigger question is:
for negative PCR control, if there is no band, many people said that I have to sequence it and delete it from other samples.
Then I have to make the negative control at 4nM and pool it to the library.
So, if I have a low negative control then amp it up to 4nM, isn't is a bias?
And when I delete it from other samples, if my sample has some of that OTU in it as a normal, so I delete it, isn't it a bias too?

I would like to make sure that I do not have a contaminant in my sequence results.
But I cannot answer myself about this question too.
Hope I can find some answers here.
thank you very much in advance.

Last edited by trong.wised; 03-08-2017 at 08:39 AM.
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