Our PCRs on β-tubulin for fungal DNA used to work perfectly. Sadly, it stopped working. A positive and negative control were included (the positive control works), all the materials checked and replaced with freshly prepared ones including the primers at the working concentration.
The PCR conditions, primers and concentrations are standardized and all the PCRs performed in the same cycler.
Any ideas on why this happened and how I could solve the problem please?
Thank you in advance!
Could be problem with the quality (DNA shearing during extraction) or quantity (very low amount or very high amount) of DNA from the experimental sample, have you determined these ? If these are fine, it could still be that some inhibitors might have come through during DNA extraction thus inhibiting the PCR..Other than this, the PCR conditions should have worked fine under standardized conditions.
I agree with Mitesh Shrestha it does seem like the dna is the problem. I would run a range of dna concentrations in the pcr starting with less than you are using now to up to 4 times as much in case of inhibitors and meanwhile check for any differences in the reagents of the dna preparation method for old reagents ( old phenol is particularly poor in phenol chloroform preps for instance. try re precipitating a non working sample in salt/ethanol to see if the resulting dna works any better. Check the od 260/280 and 230 against that of the working positive control. Some inhibitors absorb at 260 so you think that you are adding a lot of dna but actually it is much less than expected. Although the control sample works it is possibly worth trying another taq as some enzymes are less fussy about contaminants than other enzymes
I worked with fly DNA isolated from fresh as well as preserved by drying specimens some time ago. For me the problem was the poor quality of DNA in some cases (fragmentation) and presence of inhibitors despite usage of dedicated kits. Fragmentation may cause lack of products despite high DNA ammount estimation. I found two solutions for the contamination problem. One mentioned already by Paul Rutland, dillution of DNA matrices (I used 10x dillution) or more expensive one purification of DNA by Ampure (or analogs of it). Addition of BSA to PCR reaction helped as well as it binds inhibitors. Furthermore usage of Phusion polymerase instead of Taq helped me in some cases.
Is your DNA set up in slave plates or single-use tubes? It may be that you've freeze-thawed your normalized DNA too many times. We create stock plates at extracted concentration, normalized plates to the concentration we use in amplification, and then slave plates with only the amount we need. This was a protocol we started once we had the exact same problem you're having.
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