Bit of a puzzling scenario here for some samples...
DNA samples extracted from water using BioBasic soil kits, will amplify 16S rRNA(~400bp), and ~700bp region of COI gene very well.
However, the same samples will not amplify COI regions of 300-400bp, which is my target.
PCR negatives and positives work all the time. Tried temperature gradient, new reagents, diluting the samples (1:3, 1:10 dilutions), loading 2.0ul of template (typically load 1.0 or 0.5 and both don't work) and double PCR but no results for that size. I have had issues with inhibition before but wouldn't inhibition affect both primers?
Why will it not amplify a shorter target, but will work for a larger region?
Thanks!
Your primer design is based on published genome database, isn't is? If you are unlucky and have mismatch at the end of your primer with real sequence you never get your product. Try to redesign primers for region a bit of upstream of forward primer and downstream of reverse one.
The COI800 primers are from published papers. The other primers have been designed and tested on controls which work well at all dilutions and at various annealing Ts. I have used the same primers on another set of water samples (but taken from a clean tank, without soil and organic matter - extracted the DNA from the filters the same way and the primers worked perfectly). Can't seem to overcome inhibitors in my other samples.
I get amplification only when I do a double PCR, so in total has to be around 55 cycles. 35 cycles first run, use that PCR product for a second run - I get some samples with my product but then I get a lot of unspecific bands as well. I've used different amounts of DMSO, betaine, diluting the samples, and up to 40 cycles. Seems to only work when pushing it past 50.
Hey Katherine,
Are you putting both the primers in the reaction mixture?
I put the primers in diff PCR reactions. I tried a nested PCR as well but I got my.800bp band and then lots of smears. Controls worked every time.
These primers are species specific and work well at diff template concentrations. I have designed a few sets that work on controls..just not the eDNA samples.
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