I am struggling with a serous problem in detection of GMO by routine PCR using CamvP35 and Nos amplification primers. This is a very serious problem I am facing since 4 months in our lab.
Where I am able to see the good amplification of other than these genes. Many PCRs I did to amplify soya's lectin gene maize's inverase gene rape seed's hmgA gene, event specific detection of Mon810 by EuJRC method(by using the primers mentioned in that method) a PCR of chloroplast specific primers and many more.
I had good and uninhibited amplification by the above said PCRs.
I am not able to conclude the effect why it is coming with using GMO detection primers especially when I changed all my reagents, decontaminated lab area, ordered fresh oligos freshly isolated CRM DNAs. But the problem still exists.
Please help me anyone if you have seen this kind of PCR results before and how to overcome with this problem.
1) One possible reason may be non specific primer, so please check the primer sequence specificity
2) If the primer is specific to your targeted site, please standardize your annealing temperature
3) Try to dilute the DNA concentration(like serial dilution) then perform the experiment.
4) Run some control gene along with this reaction, so that you can get the idea about this problem.
Good luck
It seems, you managed to introduce looping into your amplicon; for that reason, the ladder looks like perfect concatamer. Is there any restriction in your amplicon, to check whether what you're seeing is indeed one fragment?
Non-specific bands in PCR usually caused by low temperature annealing temperature of primers. In this case, these bands disappear with increasing annealing temperature.
Furthermore, the pattern of bands is are seen in electrophoresis ofLAMP products under isothermal condition
Hi,
Is the effect I'm getting due to contamination caused by looping of primers i used or by external contaminant which is leading the experiment this way.
I am totally ruined by this work.
I am not able to think properly also. not in a situation to conclude anything.
please suggest me how to avoid this kind of outcome from PCR
Hello,
Please try with gradient PCR and find an appropriate annealing temperature. Hopefully you can order new primers.
Some variables I agree with from above include appropriate annealing temperature, proper primer design. Another fact which often neglected is tubes, use thin-walled PCR tubes. PCR machines have varying ramp up and ramp down cycles, and if the time of extension is just for 30 s and you are doing a large volume, then your entire reaction mix may have not reached that temperature if the plastic of PCR tube is thick. I have seen something happen to me long ago, I changed the PCR tube to thin walled rather than conventional 96-well PCR plate for 50 samples and everything works great.
I agree..check your primers and set more stringent PCR reaction like increase annealing temperature and reduce extension time
Is is possible that these primers could amplify multiple products? For example, the transformation construct could have more than one gene under control of the 35sP and nosT. Or some vectors will contain a series of 35S promoters (in a row, all to increase expression of the gene of interest), then you would see a laddering effect.
Dear Lakshmi Narayana Lavu
This is a problem of non specific amplification and above all suggestions are valuable. More stringent PCR reaction with more specific primer is the answer. However, you can try hot start PCR and you can also try additive like DMSO to get more specific product. I will suggest to go for HOT start PCR where you need to add one component either dNTP or enzyme after the denaturation step. This reduces the non specific amplification at initial cycles. I also suggest you to have a little high annealing temp. hope this helps.
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