In our lab, we are trying to do real time PCR for some genes. One of them has a size of 85 bp. Whatever we do, we had an amplification in negative control. When we run agarose gel, it always a primer dimer (it likes 2 bands together as the difference in size between the product and primer dimer is small.)
I tried to do a variety of conc and a variety of high annealing temps but no thing works.Everytime I decrease the primer conc, the CT value increases in both sample and ntc! (appear late than before).We don't have the capability of order a new one.
For melting curves, All of them are giving peaks at 70°C (which I think is primer dimer melting temp), lately it gave us melting curve peak above 80°C, but the problem is that the same melting curve peak appear in NTC and the difference between ct of the sample and ntc is about (3-4) cycles only. Can you help me?
I think your primers are not good and I recommend you to design a new primer pair. If primers are not good, adjusting other parameters would not help much. Did you test your primer pair with some samples which have worked? And what is your positive control?
It is not clear to me how many problems you have. I agree with Abhijeet Singh that the answer is to design better primers. This would solve the primer dimer problem and also if the amplimer size is larger then it will solve any post pcr contamination which may also be present ( it is hard to tell without a picture)
Primer dimer can be reduced by using a hot start polymerase if you are not already doing so. A normal end point pcr of a ntc and a working positive sample would help clarify if you have only primer dimer or also have contamination
The problem is that I cannot eliminate the primer dimer.
We did conventional PCR and also get dimer (photo in Black) (3` is NTC)
the second photo which mostly as we predict (primer dimer and the product) is shown also.
I sterilized all equipment with 10% bleach (this for any possible contamination) but nothing changes
we already used a hot start polymerase .
Marwa Hany
mage 1 is a lovely result with no PD showing. My concern is that this image was obtained a while ago when you were setting up the pcr and that since then you have contamination in the system. I assume that there is not a NTC in image 2. I think that if this is correct then you should repeat image 1 with 2or 3 ntcs and one positive samples. If the ntcs are positive then you have contamination .
In image 2, lane (3) and (6) are negative controls
In image (1), lane (3`) is a negative control
Image (2) is done before image (1)
Image (1) is end point PCR-product gel electrophoresis
Image (2) is real time PCR-product gel electrophoresis
In my opinion, your primers are not good, have you checked the primers for heterodimers and selfdimers?
Thank you Marwa Hany . since image 1 is clean and run later than image 2 it would seem that either the primer dimer is only weakly contaminating and the sensitivity of rtpcr is picking it up early which I think is the less likely possibility or that you have contamination in some aspect of the RTpcr that is contaminated which is not found in the end point pcr.....any reagent in rt only or plasticware, set up area if different, plasticware, different pipettes used in setup,pcr machine. . The quickest solution is still designing one primer outside of your amplimer if possible then the current contamination cannot amplify but replacing all reagents,cleaning all areas,equipment and new plasticware etc may be worth trying but primers are cheap
How image 1 is clean and 3` is NTC got a band?
I sterilized everything even the pipettes. I am thinking to put them under UV for 1 hour and give it another trial. And use a new syber green and still the NTC gave a signal.
The problem is not the price, it will take time to get a new primer (shipping from outside country takes a lot of time due to certain laws in our country)
One of them has 3 GC clamps (can form strong self-dimer) structure, but heterodimers have a weak possibility
With high Gc it might have been good to start using dmso to break any G quadruplex or secondary structure issues but it will not help the situation now as you already have the problem. If image 1 is clean and image 2 is contaminated then because image 1 is a result after image 2 it is likely that something unique to the qpcr is where we find the contamination. Sterilising does not destroy dna it just kills bugs but mainly the dna is still there and remains a problem.
Initially I would use another workers area away from yours. Use their pipettes and plasticware and use all new reagents ..ideally their pcr reagents and a new sample of your primers. If the result is clean results then strip down your pipettes and clean them, clean your area and use a new set of plastic tubes /strips/plates. Remember that water as a reagent can also be contaminated. Sometimes you can get contamination by airosol when opening tubes or by wet gloves transferring running buffer back from agarose gel tanks into your working area.
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