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PCR inhibitor in template DNA


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#1 aparis09

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Posted 21 February 2014 - 09:48 AM

I've been attempting to isolate 16S rDNA from bacteria grown on contact lenses for several months now but always seem to get some sort of inhibitor when I run PCR. My positive control works great (just E. coli DNA) but when 1 uL sample is added to the control I get no amplification. I have very little sample left but have a good concentration for approximately 150 ng/uL based on my nanodrop-1000. I've tried dilutions down to 1:1000 for the samples and still get inhibition. We've also used several DNA cleaning columns and alcohol precipitations to try and get it out but no luck. Any ideas?

 

P.S. my 260/280 ratios are 1.84 and 260/230 is 1.32

 

Thanks for your help!



#2 phage434

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Posted 21 February 2014 - 04:48 PM

This suggests to me that either your primers are not working, or that your cycling conditions are wrong. Could you tell us (1) what species you expect (2) what primers you are using (sequences) (3) what your cycling conditions are (4) what enzymes, dNTPs, etc. are used including amounts. Have you looked at this page and the references:

http://openwetware.o..._identification



#3 aparis09

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Posted 23 February 2014 - 09:48 PM

Phage, I expect a range of species as I'm doing a culture independent analysis. Most-likely looking at staph. epidermis and strep. pneumoniae. The primers I am working work great for isolated E. coli DNA that I'm using as my positive control, I'm using modified 519F, 1492R primers with an EcoR1 site built in. The actual sequences are 5'-ATGCTTGAATTCCAGCMGCCGCGGTAATAC-3'  and 5'-ATGCTTGAATTCTACCTGTTAYGACTT-3'. And like I said before, I am getting robust amplification of my positive control. I've tried varying cycling conditions but the reference I have for amplifying low-yield DNA with these primers is http://mic.sgmjourna...1/3305.full.pdf and it calls for a rather unusual PCR protocol. I've also tried a standard protocol optimized for the NEB Taq Polymerase and Thermopol buffer I am using. dNTPs are at a 200uM each.

 

I've tried cleaning the DNA with several kits and nothing seems to help. Literally no product at all and I'm running out of time. I need results soon or have to call it quits. Thoughts?



#4 phage434

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Posted 23 February 2014 - 10:12 PM

That reference shows annealing temperatures of 45, which almost never works for me. Try 55 instead. Also, I don't believe 25 cycles would be enough. Try 35. I'd suggest a master mix rather than separate components, which simply gives you an opportunity to mis-pipet or confuse yourself with little or no advantage. I think you were on the right track in using high dilutions of your template.

 

Your second primer looks wrong. First, the binding region for the template is too short, only 15 bp. Second, it has the wrong sequence:

          TACGGYTACCTTGTTACGACTT -- 1492R

ATGCTTGAATTCTACCTGTTAYGACTT -- your primer

                  GGTTACCTTGTTACGACTT -- From the paper you linked

It's missing a second T after the TACC. I suspect you'll have trouble with this primer.






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