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Old 09-23-2013, 06:42 AM   #1
Elara
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Location: Germany

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Default 16S rDNA PCR - HIFI Polymerase whithout Microbiome?

Hi,

I'm doing 16S rDNA Ampliconsequencing on our FLX+. I'm trying to generate my amplicons by using Roche FastStart HIFI Polymerase. My negative control (only mix and water) is always positive. But you can only see this if you take all your PCR-Product and run it on a Agarosegel. We also sequenced this negative controls and found a total microbiome. I also found a paper (The evidence of contaminant bacterial DNA in several commercial Taq polymerases; Romanian Biotechnological Letters, Vol. 18,No.1,2013) where this problem is shown.
So, does anyone know a HIFI polymerase which is "pure"?
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Old 09-24-2013, 05:47 AM   #2
trexbob
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I've had good luck with NEB's Q5 Hot Start Hi Fi 2x Master mix. My negative control PCR's are always negative. Are you using filtered pipet tips and quality molecular biology grade water?
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Old 09-24-2013, 06:12 AM   #3
Elara
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Default changed everything

Ok, thanks. I'll try that.
I've changed everything, odered new primer, use filter tips, sterile molecular biology grade water. I even made my PCR under a laminar flow. Just to test if something changes.
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Old 09-25-2013, 05:44 AM   #4
hewei
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Be caution your primer, we found prime maybe the contaminator source.
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Old 09-25-2013, 06:17 AM   #5
cliffbeall
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We have used Accuprime II with success.

I would also caution against running too many cycles - you probably want to stay at 30 or below.

You are hopefully amplifying experimental samples with tens of thousands of copies of 16S - that's an assumption of the experiment.
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Old 09-26-2013, 01:13 PM   #6
Isequencestuff
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We use Enzymatics VeraSeq 2.0 HiFi "ultra pure" Polymerase. Our negative controls are always blank. Its a seems to be a pretty pure enzyme although it's not a hot start, for that feature I'd go with KAPA HiFi.
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