I want to use one step RT-PCR to amplify human RB1 gene (mRNA-->cDNA). When I looked at NCBI nucleotide database (NM_000321.2), the gene has CDS on it. Which one should I amplify, the complete mRNA (4.7 kb) or CDS only (2.7 kb) ?
NB: I want to clone the gene to plasmid vector (vector size: 4.7 kb) in E. coli, then express the gene into protein
If you just want to test its expression by RT PCR, design your primer pair close to the 3' end, 300 to 500 bps will work well. If you want to express the protein, CDS amplification is needed. In any case, you will not need the whole mRNA. The 5' end of mRNA is likely lost on many if not most during your preparation.
if you want to isolate the gene and you know the CDS sequence then that is enough to isolate CDS and clone as according to your upstream work. you do not need to go for mRNA isolation till you do not need specifically for your upstream work.
Hi Afdilah,
You cannot use the entire mRNA sequence to express in e. coli. You need to include a Shine-Dalgarno box (ribosome binding site) a few nucleotides before the initiating ATG codon, so the 5' UTR will block expression.
Plus, it is a big fragment to amplify (I highly recommend NEB Q5 polymerase if you must), so I would investigate whether places like Dana Farber has it already cloned for you (Gateway system, using pET62 DEST (discontinued, but ask me if you need it)). You probably need an affinity tag for purification, so a version without a termination codon for a C-term tag might be needed.
For cloning you just amplify the CDS region.But remember SD sequence is necessary for ribosome binding.
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