Posted 09 January 2011 - 07:10 PM
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.