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Old 04-14-2015, 06:17 PM   #1
Location: South Korea

Join Date: Feb 2011
Posts: 31
Default Reducing contamination in 16s PCR for metagenomic library prer

Hi everybody,

Recently i came across with the contamination with he PCR kits while i was trying to amplify the 16s Amplicons using V1-V2 primers. i have used Takara and Bioneer kits. But both of them gives a very prominent band even with the negative controls(primers only). i did the capillary sequencing for the negative bands, which showed the presence of some uncultured strains and e.coli.. etc....

I even tried treating them with Sau3A1. But then after de-activating Sau3A1, the taq polymerase becomes inactive....

Can anyone suggest me a good kit for the 16s Amplicon sequencing...

Please share ur experiences if anyone came across with such problems

Thank you

roshanbernard is offline   Reply With Quote
Old 04-14-2015, 11:35 PM   #2
Senior Member
Location: US

Join Date: Dec 2010
Posts: 452

Decontamination with Arctic Enzyme nucleases might be an option.
luc is offline   Reply With Quote
Old 04-15-2015, 08:05 AM   #3
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Location: California

Join Date: Jul 2014
Posts: 198

We've seen contaminants in many kits and reagents. You can also try irradiating whatever PCR water you use...

Another option is to sequence negative controls and computationally "subtract" them from your true samples (for example, using SourceTracker).
fanli is offline   Reply With Quote

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