Trying to amplify the 3' end of Prolyl endopeptidse gene from cDNA.. need su - Hi, I'm trying to sequence a gene from the cDNA of an insect (Jan/09/2011 )
I isolated total RNA (from E. integriceps insect) n enriched for mRNA. Then, I made cDNA. My objective is to clone a specific gene (cDNA) into pGEM T Easy plasmid, get it sequenced, transfer the gene into another expression vector and express the protein in bacteria. I am looking for the sequence that codes for a catalytic triad (which is located near the 3' end) which is vital for the activity of the protein. The expected gene length is ~2,300 bases (compared to its closest match). I could not get my degenerate n gene specific primers to work beyond 1900 bases. I have been trying to get this done for almost an year. Please suggest if you guys have any suggestions to get the 3' end sequenced.
For the expression of your desired gene full length gene isolation which is very specific with 3' and 5' SMART RACE kit provided by various firms is useful.
aashu on Mon Jan 10 06:53:05 2011 said:
For the expression of your desired gene full length gene isolation which is very specific with 3' and 5' SMART RACE kit provided by various firms is useful.
I made the cDNA using the Clonetech SMARTer RACE cDNA synthesis kit for 5' and 3' end amplification. But.. that worked only for 5' end.
