Hey,
i am currently struggling with a positive non-template control in my 16S PCR to amplify bacterial DNA isolated from stool and skin.
I used new reagents, tips and tubes, cleaned all surfaces pipettes with UV and DNA-decontaminant (DNA-Exitus Plus). To amplify my DNA I used
515F (5’GTGCCAGCMGCCGCGGTAA3‘) and 806R 5’GGACTACHVGGGTWTCTAAT3‘) primers and the buffer and polymerase were purchased from TaKaRa (http://www.clontech.com/MR/Products/PCR/High_Yield_PCR/Ex_Taq_DNA_Polymerase). I used the following cycling conditions:
a. 94°C 3 minutes
b. 94°C 45 seconds
c. 50°C 60 seconds
d. 72°C 90 seconds
e. Repeat steps b-d 35 times
f. 72°C 10 minutes
g. 4°C HOLD
(see Earth Microbiome Project: http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/.)
Do you have any suggestions on how I could prevent contamination and false positive results in my setup? Attached is also a picture of a recent gel.
I would appreciate any feedback in order to improve our method.
Thank you very much in advance!
Best Nadine
Thank you for your response.
Regarding your suggestion to reduce cycle numbers and extension times, I will defenitley try to do that, in order to avoid unspecific amplification. For your information, the original thermocycler conditions were adopted from the Earth Microbiome Project: http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/.
I will also try shorter running times to reduce smear, since my expected amlicon should be between 300-350 bp.
I really appreciate your input, but in my opinion this platform should be used to also get in contact with researchers and experts outside of my own University.
Kind regards,
Nadine
Thank you for your suggestions. I followed your example and also rephrased my question, in order to present my problem in more detail.
Yes, we are planning to do sequencing in the future, but since we are clearly struggling with our PCR-setup, we have to improve that first, in order to get a better representation of what is going on in our samples as well as the efficacy of our DNA extraction method.
But still, thank you for your input, I will try and let you know.
As mentioned, the reasons for the bands are due to reagent contamination. You can treat your Taq with a restriction enzym or with DNase I prior to using in PCR. Or propidium monoazide to crosslink DNA in the Taq prep. Also, be careful when extracting DNA as many (most?) extraction columns are also contaminated
Thank you for your suggestions! I am currently waiting for all the new reagents to be delivered and hope that this will help me get rid of the contaminant.
I was aware that reagents in commercial extraction kits have been shown to have their own "microbiome", but not that the columns can also be a source of contamination One would expect them to be sterile in an kit sold to be for microbial DNA extraction. Would UV treatment prior to use rule out the problem in your opinion?
Best, Nadine
Hello,
Maybe you could try a different polymerase to check if it's due to the reagent. The GoTaq from Promerga for example (not a low error rate and expensive polymerase, it's just to check). Maybe try a different melting temperature, a gradient from 50 to 60 °C to find the best temperature. Make a negative control with just water. For all your preparation/dilution/etc, use tri-distilled water (or commercial RNA-free water).
Best regards.
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