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neja 02-27-2013 05:12 PM

polyT tailing
 
Hi

Has anyone ever tried adding a poly-T tail to your PCR primers to construct a functional gene? In a way I want to artificially construct 'cDNA' and insert it into a MCS of my plasmid which will be later used for homologous recombination and chromosome tagging.

I'm asking this because I have my gene of interest on a lentiviral plasmid. It is directly linked to a gene that I don't want to have in my construct by T2A sequence. SV poly A signal is quite remote. Bringing SV polyA close to the gene would be quite laborious and there are also WPRE sequences for which I don't know how would they influence gene expression if they are inserted via HR.

My idea was to construct the functional gene (promoter, operator, gene with start and stop codon, polyT) by PCR and insert it into another plasmid which I'll later use for homologous recombination. I'd insert the poly-T in the reverse primer. A restriction recognition site will follow the poly-T which will be later used for cloning.

Has anyone ever done this and it worked?
How long was the poly-T tail?

Thank you,
neja


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