I extracted total RNA from blood leukocytes and produced cDNA from 2ug of RNA, which assessed cDNAs integrity and annealing temperature of primers by electrophoresis.
In following steps, for RT-PCR i added 12.5 ul 2* master mix (sybr green) + 1.5ul cDNA + 0.75ul forward primer + 0.75ul reverse primer + 9.5ul sterilized water and mixed them very well. but i did not get any curve in qRT-PCR.
How could i troubleshoot. i assume that one of below conditions may interfere.
1. I wrote samples number on the cap of strips (e.g. 1, 2, 3, 4, ...). does it reduce the fluorescence absorption?
2. my master mix is Amplicon 2* sybr green. But it is expired about 1 year. does it have effects on results?
Hi Farzin
you should also test your primers by in silico PCR analysis.
try the UCSC website (http://genome.ucsc.edu/cgi-bin/hgPcr) or the NCBI one (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome).
all the best
fred
Hi Farzin Beglari,
Yes, the first supposition, writing on the caps (to avoid) may interfere with light excitation and fluorescent signal reading, especially for Cyclers where the lamp is at the top of the thermoblock such as BioRad, ABI...
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