Hi,
I am facing problem of variable results in pcr. Despite the fact that I am adding each reaction component very carefully, I do face the problem again and again.
I checked properties of my primers and found that some of them are self annealing. Is this the reason of my problem? I am sharing one set of my primers. Can someone please check and suggest me possible remedy?
Primer F: 5-GGGGTCAAGACTGATTGGAT-3
Primer R: 5-TTGTTGTACAGCCGACACAG-3
Regards.
Hi Muhammad,
I checked your oligos and found some primer dimers (see attachment). This should not cause a problem. However, I found that the annealing temperature of your oligos is rather low. You should get reliable results if you run the PCR at 52°C annealing temperature. Furthermore, I think that you are trying to amplify Oryza sativa NAC1 transcription factor-like mRNA, and the PCR product will be around 100 bp long, which is rather short. Hence, if your oligos are not intron-spanning, you risk to amplify genomic DNA which may contaminate your mRNA preparation and may explain why you get varying results if you amplify different mRNA preps... Finally, the forward primer also binds to NAC19 (100%), and the reverse primer to NAC20 (100%). Thus, it seems as if your oligos are not specific enough... Please let me know this helps.
Kind regards,
Jochen
Yes primer self annealing can cause variation in PCR result.
There can be many reasons for getting non-specific binding in PCR .So you can
Increase annealing time if the non-specific products are shorter than your target. If they are longer than you target, reduce the annealing time.
Use less DNA template.
try these and you will get results
Hallo Hardik,
Thank you for reply. Actually, the main problem which I am facing is that my target genes are highly sensitive to Cd stress but in my case, I am getting very weak response. I don't know why?
Hi Muhammad,
I checked your oligos and found some primer dimers (see attachment). This should not cause a problem. However, I found that the annealing temperature of your oligos is rather low. You should get reliable results if you run the PCR at 52°C annealing temperature. Furthermore, I think that you are trying to amplify Oryza sativa NAC1 transcription factor-like mRNA, and the PCR product will be around 100 bp long, which is rather short. Hence, if your oligos are not intron-spanning, you risk to amplify genomic DNA which may contaminate your mRNA preparation and may explain why you get varying results if you amplify different mRNA preps... Finally, the forward primer also binds to NAC19 (100%), and the reverse primer to NAC20 (100%). Thus, it seems as if your oligos are not specific enough... Please let me know this helps.
Kind regards,
Jochen
Hallo Jochen,
Thank you very much for always being supportive. No, I am not using hot-start. With the mentioned set of primers, I am trying to amlify NAC6 (expected product size 122) from Oryza sativa and I am using 58C annealing temp.with 34 number of cycles.
Greetings
Ansar
Hallo Ansar,
I would reduce the annealing temperature to 52°C. Also, please take into account that a NCBI BLAST search also identified NAC1 as a perfect match for your oligos; the PCR product has the same size as amplified NAC6. I am not an expert in Oryza research and don't know whether NAC1 and NAC6 are two different gene products that are expressed together in the same cell, which can also explain varying PCR results. My suggestion would be to design gene product-specific oligos that are intron-spanning in addition, and repeat the experiments.
Best,
Jochen
Annealing temperature is the key factor. If you have already tried your hand on optimising altering the annealing temperature the next option would be to design/ consider more specific set of primers. Hot start PCR would be beneficial.
All the best.
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