Hi all,
I am facing the problem with the amplification of the non-template control (NTC) towards the end of the qPCR cycle (SYBR Green based qPCR). I ran 40 cycles and the NTC amplification always occur at 38th cycle onwards.
I used the same reagents / master mix for different primers and it worked well with no NTC amplification. Could the NTC be caused by primer dimer because I only faced this problem when this specific primer pair is used.
Tq.
I will agree with Florian C Priller
that this may be a primer dimer issue. Also attempt to re-design another primer set. The way you have described this does not appear to be a contamination issue... You may also run a gradient assay to determine the optimum temp for this primer set.
I agree with Laurence. Check with your product melting temperature. Also could run a gel. If you do not see your product there, and only primer dimers, it can be ruled out I think.
Thanks all for the valuable inputs! I have asked several experts in my place and they also advised me that these amplification can be ignored.
To confirm they are primer dimers, after amplification perform a melt curve analysis at the end of your program. This can be 65 to 95 degrees with an increment of 0.5 degrees for 5 sec. Melting curve analysis can identify both the desired PCR product and primer dimers by their unique melting peak behavior. Pure, homogenous PCR products usually produce a single, sharply defined melting curve with a narrow peak. Since they are smaller, primer dimers usually melt at a lower temperature than the product and have broader peaks (
See the attached file on the melt peaks of a real pcr product (on the right) which will always be above 80 degrees and dimers or non-specific PCR products that is usually smaller in size and has a lower melting peak than the desired PCR product
Zhi Xiong Chong I have similar trouble in PCR. I used a pair of primers which targets the fragment about 150bp. When I use this pair of primers, there always have NTC amplification(35 cycles). But when I change the primers with the same reagents / master mix, the NTC amplification disappears. Can anybody help me?