Hi
I will be happy for your help.
I'm doing real-time PCR with two primers called MT-TL1 and 18S, for mtDNA and houskeeping gene, and I got positive control on both NCT. I tried also different primers: ND1 and B-Globoin but also in them I'm getting positive ct value on ND1 gene (mitochondrial DNA gene), the b-globolin gene was good (undetectable).
I checked my reagents and they are good, there is no contamination. On these 2 last primers shouldn't be a primer-dimer problem.
I'm getting values of: 36.95, 35.59, 35.49
I used 10uM primer conc.
Thanks.
You have contamination OR primer dimers (or both). Run out some of your samples on a agarose gel to tell the difference.
Katie A Burnette is right. Run a gel then you may see design new primers or what!
Hi Linoy Israel
could you first test your primers in in silico PCR as in UCSC (http://genome.ucsc.edu) or NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) just to be sure you're targeting the right positions?
fred
Hi Linoy Israel,
Some suggestions to you:
1. The Ct values over 35 seem too high for me. Far away from the serial dilution curve --> results are less and less reliable. Solution: Increase the amount of template.
2. The melting temps of your unexpected PCR products are around 77oC which is way too low for a normal PCR product (100-200 bp), so apparently these are primer-dimers. Best way to check: run the PCR product on agarose gel, you should not see any band. Solutions: reduce the amount of primers to be used, check the quality of the primers using some online tools (e.g. https eu.idtdna.com/calc/analyzer ) at least the primers should be good theoretically; design new primers if needed; change SYBR may help sometimes,
Good luck!
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