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Reducing contamination in 16s PCR for metagenomic library prer


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4 replies to this topic

#1 roshanbernard

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Posted 14 April 2015 - 05:27 PM

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

Rosh


#2 phage434

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Posted 16 April 2015 - 04:37 PM

Getting no-template controls for 16s PCR is probably almost impossible. Everything has some bacterial DNA, including, likely, you PCR reagents, water, tubes. But the amounts are very small, and are overwhelmed by the presence of a real template. If you can do qPCR, you will likely see that ampllfication happens only after many cycles. I would not worry about it. If you are concerned, sequence the no-template control band, and conclude that it is different from your template sample. To really pin it down without qPCR, do serial dilutions of your template and sequence the band from the dilution series.



#3 roshanbernard

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Posted 16 April 2015 - 04:46 PM

Getting no-template controls for 16s PCR is probably almost impossible. Everything has some bacterial DNA, including, likely, you PCR reagents, water, tubes. But the amounts are very small, and are overwhelmed by the presence of a real template. If you can do qPCR, you will likely see that ampllfication happens only after many cycles. I would not worry about it. If you are concerned, sequence the no-template control band, and conclude that it is different from your template sample. To really pin it down without qPCR, do serial dilutions of your template and sequence the band from the dilution series.

Hi phage,

 

Thanks for your response. well, i did sequence the negative bands. And the blast hit varies from pseudomonas, e-coli and some unclultured etc. The main concern is i am dealing with Metagenomics using 16s Amplicons using Iontorrent. Hence if there is even little contamination, i believe that will be amplified. some one suggested me articzymes pcr de-contamination kit. However that works best with qPCR. anyays i am going to try with it.  i hope i can get rid of contaminations.

 

Thanks again for ur reply

 

regards

 

rosh



#4 phage434

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Posted 16 April 2015 - 04:58 PM

Yes, of course it will be amplified. You will see (with low probability) a 16s contaminant in your ion torrent data, but this is just the reality of your sample. It actually does have some of that template in it. The fact that you are seeing different, amplified 16s sequences reinforces my point that you are amplifying contamination -- not a real problem with you setup. If you  want better looking results, you could try a smaller number of cycles in your PCR. If I am right, then a 20 cycle PCR will likely amplify your samples, but fail to amplify your contamination. And you'll get good lookng NTCs. And sleep better at night.



#5 roshanbernard

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Posted 16 April 2015 - 05:03 PM

yeah.. i think i will try reducing the PCR cycles and try it out. For the moment i use 34X... better i reduce and see the difference.. thanks again.. :)






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