My fellow Academic colleagues!
I together with my lab mates have a PCR-related issues that we hope that some(one) of you might have encountered and hopefully solved.
In “short”, our initial PCR (MiniAmp Plus thermocycler) and electrophoresis protocol works like a charm – the latter somewhat modified. We obtain weak to strong band that yielding concentrations of 9 to 20 ng/µl following clean-up using the QIAGEN QIAquick PCR (& Gel) purification/Cleanup Kit (with an acceptable A260/A280 ratio). We obtain rarely, but from time to time, a positive electrophoresis confirmation. But as we are using the same protocol for the confirmation, as for our initial PCR, we should have no issue confirming our results (one band per week).
Usually, when we try to confirm our cut-out electrophoresis bands, running a PCR on our cDNA, something fails. We utilize the same primers and protocol, as for the initial PCR, but nothing shows up in our gel, our at best a streak. We’ve tried renewing our primer mix(s), new isopropanol, new buffers, using both RNAse-free water and the included buffer, modifying temperatures (thermocycler), number of cycles, and using the original non-modified protocol. But nothing results in an electrophoresis band when we try to confirm our initial band.
Thank you for your insights and help!
// Eriksson et al.
when you are reamplifying band remember that 10 pcr cycles is 1000 times more pcr product so 30 cycles is 1,000,000,000 times more product and that is so much that the pcr fails and you just get a smear of joined up product molecules. try diluting the initial pcr product 1/1000 in water and amplify 1ul of this using 12, 14, 16,18 and 20 cycles and you should get an amplified band at a couple of cycle numbers and probably a smear at the higher cycle numbers
Hood evening , If I understand correctly, your initial PCR is ok, do you extract it from the gel or do you test it again on your cdna, is the answer negative? If this is the case, it is clear that your genome is not stable in the storage buffer and will be destroyed, which may be due to not using dnaase rnase free equipment or your freezer is not at the right temperature. Regarding the extraction from the gel, if you have a band on the gel and even have light absorption, your DNA may have been destroyed during the extraction process. My suggestion is to start the process on a positive sample from the beginning and after electrophoresis and being positive from the gel. Extract and immediately PCR, and see what the answer is. If it is positive, there is a problem with the storage process. If it is negative, there is a problem with the extraction process.
So you're telling me that you generated cDNA from mRNA using reverse transcription primers, and then used those same primers in an attempt to amplify the cDNA?
Hi and thank you all for taking your time answering our question.
Paul Rutland: we have tried using multiple cycles; from 20 to 40. The issue is that we cannot confirm our cDNA following gel extraction, irrespective of number of cycles.
Hamidreza Mollaei: yes your description is correct: PCR and electrophoresis works, but we seem to lose our product during either gel extraction or during PCR confirmation. We store our gel cube at -20°C (usually < 14 hours).
Our gene product is about 2000 KDa.
Something degrades our cDNA - unknown what.
As per your suggestions, we've completed the whole process in one go and with similar results.
Hieu Nguyen: We extract our gene of interest from biological samples. Then using the PCR protocol from QIAGEN, we amplify our product of interest, confirm its presence using electrophoresis. Positive gel samples are extracted (100 - 250 mg) and processed as per a slightly modified QIAGEN QIAquick PCR (& Gel) purification/Cleanup protocol. A subsample extracted from the gel is then re-analyzed and confirmed using the same PCR protocol.
We currently suspecting that the longer run PCR (confirmation) might be incompatible with our product. We are trying different protocols in order to (hopefully) achieve confirmation.
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