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Trying to amplify the 3' end of Prolyl endopeptidse gene from cDNA.. need su


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#1 Yandamuri

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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.

#2 aashu

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Posted 09 January 2011 - 10:53 PM

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.

#3 Yandamuri

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Posted 10 January 2011 - 11:16 AM

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. :(




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