Truncated cDNA?

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Thu Aug 31 05:52:23 EST 2000


In article <39AE23F0.6913B6CD at sgen.slu.se>, Mathieu Ingouff
<mathieu.ingouff at sgen.slu.se> writes
>I am amplifying my gene by PCR using cDNAs.
>I do the RT with a d17-Adapter and then amplify with one specific primer
>and the adapter primer.
>And i get a single band that would correspond to a truncated amplified
>cDNA. The expected size if 3 kb and i get a 2kb band.
>I tried again using DNA prepared from a cDNA library (RT was done with a
>d17 primer)
>Same result.
>
>Actually i don't know if it is truncated but my gene belong to a highly
>conserved family and this protein would miss one conserved domain but
>well i cannot get it......
>
>DO you have any idea how i can really be sure that i have the full
>length?

What happens if you do the RT using an oligodT primer then do the PCR
with two specific gene primers rather than an adaptor and one specific
primer. Likewise do two internal specific primers PCR off your existing
PCR product. If not you probably have the wrong product. 

The only way to really find out what you have is to sequence it. 

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






More information about the Methods mailing list