Hi there,
In my experiment I need to do PCR with below DNA and Primers, I also use RT-PCR.
But I don't know why it cant be amplified. The other information is available in the attached picture.
dsDNA:240bp
TTCGCGAGATCTATGAATAAAGACACACTGATCCCTACAACTAAAGATTTAAAAGTAAAAACAAATGGTGAAAACATTAATTTAAAGAACTACAAAGATAATAGCAGTTGTTTCGGCGTATTCGAAAATGTTGAAAATGCTATCAGCAGCGCTGTACACGCACAAAAGATATTATCGCTGCATTATACAAAAGAGCAACGTGAAAAAATCATCACTGAGATACGTAAGGCCGCATTACAA
S1-Primer-F: 22bp
TTCGCGAGATCTATGAATAAAG
S3-Primer-R: 22bp
TTGTAATGCGGCCTTACGTATC
Hi Karimi --
Whenever I design primers, I like to run them through a site like OligoCalc or Benchling for analysis.
Looking at your primers through OligoCalc there seems to be two issues. First, the melting temp of your Forward primer is about 56*C and your reverse is about 59*C. Your PCR program is set to 59*C for annealing, and this is too high for the F primer. Always set your annealing temp a few degrees below the lowest melting temp for a primer. In your case, i would set it to 51*C instead of the 59*C you currently have.
Second, and probably less of an issue, is that your Reverse primer has a potential hairpin formation (check Oligocalc-Self Annealing) which could be inhibiting annealing.
First try adjusting your PCR program with a lower annealing temperature. Let me know if you have success with that! Good luck.
http://biotools.nubic.northwestern.edu/OligoCalc.html
benchling.com
Hi Karimi,
how does your gel look like?
Are there any bands, or is it empty?
Best,
Konstantin
Hi Karimi --
Whenever I design primers, I like to run them through a site like OligoCalc or Benchling for analysis.
Looking at your primers through OligoCalc there seems to be two issues. First, the melting temp of your Forward primer is about 56*C and your reverse is about 59*C. Your PCR program is set to 59*C for annealing, and this is too high for the F primer. Always set your annealing temp a few degrees below the lowest melting temp for a primer. In your case, i would set it to 51*C instead of the 59*C you currently have.
Second, and probably less of an issue, is that your Reverse primer has a potential hairpin formation (check Oligocalc-Self Annealing) which could be inhibiting annealing.
First try adjusting your PCR program with a lower annealing temperature. Let me know if you have success with that! Good luck.
http://biotools.nubic.northwestern.edu/OligoCalc.html
benchling.com
I assume you're using Taq? Or is it a different enzyme?
If Taq, I definitely agree with Sam. I think you're running this PCR too hot. I would drop the annealing temperature to 55 degrees at most.
Dear Sam Moxed,
Thank you for your attention, that's good point let me i try with it.
Dear Govinda Sharma,
Thank you, as also dear Sam mentioned, I will try with lower temperature to see what would be happened.
It's a good point to try lower temperatures, but there might be another problem. (In my opinion there should be a smear if the annealing temp doesn't fit to good.)
To test if there is another problem, try another pair of primers (maybe primers for household genes) on the same cDNA/RNA sample.
Hi Ali, the annealing temperature change by using oligocalc site. You can also change the amount of dna extraction. Finally, if the result did not! You can use another primer.
I changed the temperature (51*C and 55*C) still the Gel is blank.
I designed new primers:
F1 5'- TTC GCG AGA TCT ATG AAT AAA GAC -3'
F2 5'- TTC GCG AGA TCT ATG AAT AAA GA -3'
F-current 5'- TTC GCG AGA TCT ATG AAT AAA G -3'
R1 5'- TTG TAA TGC GGC CTT ACG -3'
R2 5'- TTG TAA TGC GGC CTT ACG TA -3'
R-current 5'- TTG TAA TGC GGC CTT ACG TAT C -3'
here is some comparisons between them in my mind the F1 and R1 may works well, do you have some other idea?
It seems like F1 and R1 could work well together. I would set your annealing temp to between 48*C and 50*C.
Do you have a positive control you could run with your PCR? Are you sure the primers are an issue and not the other components in the reaction? Perhaps it would be useful for us to see what reagents you are using for your PCR reaction.
Dear Samantha,
I appreciate your kind attention.
As soon as i get the primers i will try with that temperatures.
I don't have any positive control, what do you suggest for the positive control?
For the PCR reaction I use AccuPower ®PCR PreMix. the info is here as an attachment.
Also, the other point is that i use Real time PCR, as i check in the manual and the technician visited our university he said it can be used as a normal PCR too, i don't think this may some issue in the result what do you think?
Also , I tried to check my DNA and primers in "genomecompiler" . the software highlighted some area as "BglII". does it have some effect in PCR?
Dear Ali,
as i mentioned before, you could try your PCR with a household gene as a positive control. For example ß-Actin or any enzyme from the TCA cycle. If your gel is still empty you'll definitely have a problem with RNA isolation or cDNA rev. transcription.
But if your household gene PCR works well, you can be sure that it's a problem with your primers or the amount of GOI. To figure out if it's your GOI, that is to less expressed, you could do a nested PCR.
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