Dear Heather Cv

For troubleshooting, I would like to first ask which protocol are you following. There are several kits/protocol versions for performing TRAP assay.

My TRAP assay protocol is based on this publication - https://www.nature.com/articles/nprot.2006.239

I am not certain if you are running a qPCR based TRAP protocol or observing the TRAP ladder on a gel.

I am assuming that you do not detect a TRAP ladder in the CHAPS only control.

Assuming this, then I would recommend to make sure that you are changing pipette tips EVERY TIME when pipetting the positive control, test samples and heat inactivated controls in the PCR reaction. And once again using fresh tips for loading each of the sample on the non-denaturating gel or the qPCR plate. TRAP assay is highly sensitive so pipetting has to be done with great care.

Secondly, I did not quite understand what you meant by - "I am thinking that perhaps there are telomere fragments in some of the samples so maybe I need to clean the DNA from the samples?" . In case/ even if there are telomere fragments the TRAP ladder should still not appear in the heat inactivated sample.

The heat inactivation of the sample is done before you initiate the PCR. And since the heat inactivation denatures the telomerase enzyme required to generate the hexameric product you should only see one band of the internal control primer (or if u think there is a telomere fragment then maybe a second band of that) and not a full length TRAP ladder. if you run a qPCR then you might see primer dimers but the melt curve will be different that of the TRAP positive samples. Have you checked this part as well for troubleshooting?

Best wishes

Shambhabi

Shambhabi Chatterjee I am using the Millipore Sigma S7700 kit (has primers and all reagents) and running it on a gel after PCR. Detection using SYBR green after electrophoresis. I have used this protocol with other samples and did not get this issue as much (I got primer dimers though).

No TRAP ladder in CHAPS only lane but there is a band at about 50bp which seems like primer dimer.

I change pipette tips for each sample.

For the telomere fragments, I am wondering if maybe there are DNA fragments in my sample already and these would not be degraded by the heat treat that denatures the enzyme. But they would be amplified in the PCR. So I am wondering if there could be telomere templates that the primers are elongating.

Since I am not doing qPCR, I don't have a melt curve.

I am also wondering if maybe the other bands are primer dimers and so thinking about increasing the annealing temperature. But I am not sure how much I should increase it by. Now it is 59*C. I am also already using hot start to try and reduce this.

Thanks for any ideas you have!

Dear Heather Cv

hope you have been able to troubleshoot this issue by now. here are some suggestions in continuation of the previous comments, in case you are still trying to figure out the issue.

If your samples have DNA contamination then one option might be to use serial dilutions of your sample in the assay. In case it is DNA contamination then the band should also get fainter with each dilution. In case it doesn't fade away with serial dilutions of your sample then it is definitely not coming from the original sample and more likely from the reaction master mix. ( I personally don't understand how the primers could be elongating telomere templates in blood samples. perhaps you could have a closer look at how the samples had been processed and that might explain if at all there could be free telomere templates in the blood samples)

instead of changing annealing temperature I would reduce the amount of primers that you are using in the reaction. titrating the primer concentrations can more likely help to eliminate the presence of excess primers which can lead to primer dimers.

good luck !