G/C tails in expression constructs

Ed Rybicki ED at micro.uct.ac.za
Thu Nov 12 05:14:31 EST 1992


> To:         methods-and-reagents at net.bio.net
> From:       rosswhet at forbt2.nrrc.ncsu.edu (Ross Whetten)
> Subject:    G/C tails in expression constructs
> Date:       11 Nov 92 18:08:00 GMT

> Hello netters!
> I am making an expression construct with a "constituitive" promoter driving
> transcription of a full-length cDNA that was originally cloned using G/C
> tailing at a Pst I site. Are the G/C tails likely to present problems for
> the expression of the fusion gene? There are likely to be 20 to 50 consecutive
> G residues in the 5' untranslated portion of the resulting mRNA. This is
> a plant construct using the CaMV 35S promoter, if that makes a difference.
> Comments from anyone with experience in this issue are welcome, as are any
> references to papers that address the question. Thanks.

Colleagues of mine had a similar problem with an animal virus gene that
had been cloned using the same method - they simply sequenced it, then
PCRed it out of the clone using primers specific for the 5' and 3' NCR
sequences inside the G/C tails, and recloned it into the expression
vector.  Probably safer, all round.  Hope this is of use.

  ____________________________________________________________________
 | Ed Rybicki, PhD             |    "Now you've got the hang of it    |
 | (ed at micro.uct.ac.za)        | There's nothing you can't do with it |
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