need help cloning cdna of a known protein - (Dec/14/2009 )
Hi everyone, I have found a new protein to study, but I am unsure how to proceed. This is a protein whose mRNA sequence is known and published. Theoretically, I need to get cDNA from mRNA using reverse transcriptase, amplify the cDNA I've made, and then stick it into a mammalian expression vector, important for my purposes.
I don't know how to proceed. I haven't done very much of this type of mol bio.
Do I need fancy things like RACE, or can I just use RT-PCR?
Any help/suggestions would be great.
I was going to try sticking this protein (mRNA is ~5000 bp) in pCl-neo.
pretty standard: design primers for mRNA with restriction sites, amplify one strand, use that for PCR with another primer (with a restriction enzyme), gel purify the PCR, restriction digest vector and PCR insert with the same RE, phosphatase treat vector, ligate insert and vector, transform into bacteria, pick colonies and sequence to confirm you have the insert
by the way RACE is when you dont know the sequence of mRNA.