I want to amplify cDNA for a specific RNA in plasmid, I will transfect it into mammalian cells, generating full length transcript(3kb) is necessary for my experiment.
Hi Nishu
there are some tips to follow when designing primers (as in the attachment) but the most important thing is the choice of the target. just be sure to be sure to amplify the right sequence and only this one by testing in silico your primers (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome).
fred
I think this video would be helpful for you.....
https://www.youtube.com/watch?v=TplxIHP4uHE
Besides the common considerations of Primer designing, I think the most important is that your template (cDNA) contains no any genome, or you will possibly amplify sequence containing introns.
Then you just need to get the sequence of the isoform from NCBI, choose proper restriction enzyme that will not cut coding region of you sequence, and design the primer pair at both end (translation start site-ATG and stop codon (TAA, TAG, TGA) with the cutting site.
If you want to target your protein sequence at C-terminal, do not introduce the stop codon. and be sure there is no framshift at both end by in silico.
Thank you Stephen and thank you fred for detailed information. I use to go through the same steps.
I wanted to know if there is anything I should keep in mind while designing primers to get cDNA amplified for a particular transcript isoform , I will be using it for cloning and IVT as well.
Hi Hui,
Thanks, it would help.. but what if I want to look at the role of a specific RNA in human cell lines and not just it's translated product, its both of the UTR must be included ?
And for this transcript, it's 5'UTR have some CAG expansion mutation whose role I want to look at.
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