I set up pcr reactions with primers used in amplification of telomere sequences. these primers are 35 and 36 nt in length and have 51% and 53% GC content. No template controls give ct value before samples. I tried using HPLC-purified primers, but the problem persists. What can you suggest? Thank you from now.
Thank you for your answers. I have attached the gel image and the amplification curve. As I am trying to amplify telomere sequences, I need to see a smear band for my sample due to the different sizes of telomere sequences being produced. But probably my primers form primer-dimer. I changed all the components one by one but the result is the same. Now I am trying to use PCR additives such as DMSO, BSA. I am using 20 ng Arabidopsis thaliana gDNA and SYBR mix for reactions.
Set up an agarose checker gel and run the no template control and one positive sample. If they both show bands of the right size then you have pcr contamination. The quickest solution is to design one of the 2 primers outside of the current amplimer.ie if your amplimer is 100bp then make primers for 130bp. This will solve the problem but you are doing something careless to get contamination of the new primers unless you are more careful with opening tubes and handling pcr product and electrophoresis buffer if you are running checker gels.
If you cannot design new primers then clean your pipettes inside and out,wash your working area and pcr blocks well, change your plasticware . Run your next pcr with 4 NTCs and 1 sample using both your pcr reagents and another researchers pcr reagents and use their pipettes to get an idea of where the contamination is coming from...reagents,working area,pipettes
A positive signal in the no template control (NTC) indicates contamination or primer dimer formation. Please check:
https://www.sigmaaldrich.com/MA/fr/technical-documents/technical-article/genomics/pcr/troubleshooting
Article Amplification of nonspecific products in quantitative polyme...
Probably contamination, check on an agarose gel.
Change out all components of the reaction for brand new items
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