Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

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.

-Yandamuri-

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-

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. :(

-Yandamuri-