Hi everyone for the past 3 weeks I've been battling with PCR. The extraction is mostly a success, however, 16S PCR just seems to not work. I've changed primer concentration, Nuclease-free water and the concentration of my DNA template. Please help thank you :/ conditions being as programmed to perform 35 cycles of 95• (5 min); 95• (30 sec); 53• (30 sec); 72• (1 min) and final stage 72• (10 min). PCR RR mix PCR Master mix 12.5, Nuclease-Free Water 8.5, RP 1, FP 1, and DNA template 2 microliter = 25 PCR RR MIX
Next thing I would look into is the annealing temperature.
There is a nice Tm calculator on the NEB website that also considers the choice of polymerase. The suggested annealing is rather a starting point than the actual ideal temperature.
http://tmcalculator.neb.com/#!/
Try touch down PCR. Dilute the DNA as it may contain PCR inhibitor. You can use around 10 ng-100 ng per reaction.
The programme for touch down will be
Denaturation- 94 oC- 1 min
Annealing- 65 oC - 55 oC (1 oC lower per cycle)
Extension- 72 oC – 1 min
3.25 cycle
Denaturation- 94 oC- 1 min
Annealing- 55 oC (1 oC lower per cycle)
Extension- 72 oC – 1 min
Followed by final extension 72 oC – 30 min
Hello,
DNA extracted from water may contain PCR inhibitors. What protocol do you use forDNA isolation? May be you need to try another protocol?
Hi Audrey,
When you say it's not working, do you mean no amplification or unspecific amplification?
As I can read you're trying to amplify 16S, which primers do you use, 27F/1492R? or something shorter?
As a template, 2µl, but what is the DNA concentration? are you supposed to coextract DNA from other organisms?
Someone suggested to try with 10 to 100 ng per reaction, I'd rather choose something like 1ng per reaction. beside the fact that you probably don't get huge DNA quantity after extraction, that might reduce the influence of PCR inhibitors.
In order to check things like PCR components, and set up, think about running a control with either a sample known to work for 16s or gDNA from a strain.
(to my opinion, if matermix, enzyme, primers are ok, the problem is rather connected to your samples, quality or quantity)
I concur with the comments made by David Bru. 1ng bacterial DNA should contain about 1000 copies of 16S, if not more, and that should be enough assuming good DNA quality. An extraction protocol that avoids excess DNA fragmentation is particularily important with longer amplicons (eg. 27F/1492R).
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