Lately, I am being plagued by a hit and miss situation concerning the reproducibility of my PCR. I am trying to check expression of a gene reported to be expressed in the zebrafish intestine. I have been doing RNA extraction followed by cDNA synthesis, which I use for the PCR. I am now wondering if I might have more success if I designed primers that amplified a different region of the gene of interest. It does seem odd though since I have no troubles with the consistency of the housekeeping gene amplicons. But then again, housekeeping gene expression would be expected to be more abundant and so they may work with almost any primer designed for them. Will designing a different set of primers help?
try to prepare cDNA using anchored oligo dT primers and also try to design your primers more towards the 3' end of the mRNA, preferrably within the 500 bp of the start of the poly A tail.
You need to design your primers such that they do not anneal to hairpin structures that may form in your template. To do this check for secondary structure in your template at the required annealing temperature using DNA fold http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form
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