Hi,
I am working on a gene with low expression. I decided to construct my standard curve using plasmid clones.
1. I chosed a primer set specific for that gene.
2. I did standard PCR using this primer and then this PCR product is cloned into the TOPO vector and transformed the reaction into electrocompetent E.coli cells.
3. Plasmid cDNA is purified, linearized and quantified.
4. Copy number is calculated and dilution series are prepared.
5. Then I did real time RT-PCR using same primer set which I used for preparation of cDNA clone (can I use same primer set???).
My question is, what are you thinking about my steps to prepare cDNA plasmid preparation? Can you offer any paper about that? Thank you very much.
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