Hello everyone, I’m facing an issue where my PCR isn’t working, and I’m trying to troubleshoot whether the problem lies in how I handled the lyophilized primers. Here is the step-by-step protocol I followed:
Is this approach technically sound for routine PCR applications? Are there any risks in storing the primer overnight at 4°C before preparing the working dilution?
As long as nuclease-free water is really nuclease-free, the other steps are not as crucial. Normally, diluting the primer from -20 °C before use is still okay, even without the 4oC overnight, since primers are very stable anyway. The conc. used seems okay as well. Maybe you should troubleshoot the sample integrity, annealing temperature, or primer specificity. Good luck!
Hi Diksha,
I agree with Ahmad Hafiz Murtadha - the handeling of your primers sound good ( I would say you can skip the heating step because short oligos dissolve very rapidly in water - however I think heating will not interfere with the primer integrity).
Regarding your PCR - do you have a good positive control from which you know that the PCR should work on your template?
I think one of the main reason PCRs are not working is due to impaired template (mentioned also by Ahmad). As you wrote that you are doing RT-PCR I would conclude that your primary template is RNA (I am asking because the abbrivation RT is sometimes used for Reverse Transcription PCR and sometimes for Real time PCR)?
If your primary template is RNA check the template on a gel (checking with a photometer is not enough because you will only get information of the concentration and the purity of nucleic acid but you will not now if the template is still intact).
However, if your template is DNA I still would check on a gel, because even if it is more stable than RNA it could be degraded.
Some other advice on the template - don't use to much template this might also interfere with the PCR.
If you have a positive control and it is working on your template I think your template is intact.
If the template ist intact the next thing you should look for is the annealing temperature. if you can perform a gradient PCR to see which temperature is the optimal annealing temperature.
These are the things I would definitivly check first. There are a lot of other things you can troubleshoot, but if nothing work you should get new primers.
Good luckk
Maybe you can share your PCR protocol and we can try to see from there.
Diksha Rani
Hi, Diksha Rani .
There is no problem with the operation process you provided. Because your question did not specifically mention the failure of the RT-PCR experiment, what is the corresponding problem.
For example: no PCR bands, non-specific bands, or other problems. For solutions to these problems, the RT-PCR technical documents provided by MCE have very detailed analysis.
You can directly refer to the FAQ module, in MCE's Protocol document. match the experimental problems you encounter, and find the corresponding causes and solutions.
Method [Protocol] RT-PCR (Reverse transcription PCR) Technology (Pr...
Wish you all the best, and you can continue to contact me if you have any questions.
Lucian Miles We are getting good and specific results for Beta-Actin (custome synthesized from sigma) and GAPDH (purchased from thermo) from same cDNA sample. Other primers like APOE, SERBP-2 etc (custome synthesized from sigma) are not giving any bands. However, Some of the genes are giving multiple bands.
Diksha Rani , I would understand that the problem you encountered is either the inability to amplify the gene or the generation of non-specific bands.
Because you successfully completed the amplification of other genes with the same cDNA, it means that your cDNA sample is of good quality and the amplification system (PCR enzyme, preparation method) is good. The problem may be with your primers.
If the amplification yield is low or there is no band, it is recommended to reduce the primer concentration (50μL system uses a final concentration of) and use a more efficient DNA polymerase. GAPDH has a short sequence and can be amplified relatively easily. Genes with more than 3k, such as SERBP-2, may require more efficient PCR amplification enzymes, such as High-Fidelity PCR Master Mix.
If non-specific bands are produced. It is necessary to first determine whether the gene product contains a band of the target gene size.
- If the band contains the band of the target gene and the non-specific band is smaller than the band of the target gene, it may be because the annealing temperature is too low (not lower than 65°C). It is recommended to increase the annealing temperature to improve specificity; and reduce the number of cycles to avoid non-specific amplification.
- If the band does not contain the band of the target gene, it means that there is a problem with the primer, and it is necessary to re-blast to see if other genes can be matched.
Oh, btw, sometimes for longer mRNA transcripts (3kbp), during RNA to cDNA conversion, the reverse transcriptase can slip midway, resulting in an incomplete transcript. Unless your target region is towards the polyA tail. If you are using a kit to convert, they would normally give you OligoDt primer and random hexamers. Random hexamers will allow for better conversion throughout the transcript, but the binding step, normally incubation at 25 °C for 5 minutes, is required, although some kits already include the step as well.
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