I am trying to do my first real time pcr analysis for flowering gene expression.
Does primers used in routine pcr different than primers used in real time pcr? What are the criteria for designing qpcr primers?
Are there any tips in making experimental design for use in real time machine?
If the kit suggest the protocol for 50 ul reaction, is it wise to do only 25 ul reaction assuming i also halved all the component?
Hello Dylan
please find the attachments which will give all the insights u need for real time pcr basic principles
Hi Dylan,
I used this website to design my primers based on the SNP of interest. It worked very well, if I recall, you just enter the sequence region and SNP of interest and it will give you the suitable primers.
Regarding reducing assay volume, it may work but you will have to evaluate and validate that scientifically.
Hi Dylan
in addition to above answers and known tips on how to design primers, I would say that you need that one of the primers need to be across one exon junction to be sure to avoid contaminant DNA amplification. testing your primers is possible electronically on the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome).
fred
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