Unstable or unclonable cDNA

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Tue Oct 20 15:55:08 EST 1992


In article <1992Oct19.191014.10871 at gserv1.dl.ac.uk>, yedwards at crc.ac.uk (Dr. Y.H. Edwards) writes:
> 
>      Dear Netters,
> I am writting my thesis at the moment and I need to find anyone who knows anythng about DNA which is unstable in cloning vectors.  I have cloned a region of 
> cDNA which was only detectable in an unamplified library and was impossible
> to maxiprep or subclone.  I have sequenced it directly from PCR product and now 
> am in the position to examine the DNA sequence and to suggest reasons for the 
> difficulty in retention of the fragment in a vector.  I have run a GCG 'dotplot'
> with the sequence compared to itself, but this reveals no obvious repeat regioand I have tried a 'FoldRNA' run.  although this looks pretty I'm not sure how
> much it means and also I have 5Kb to examine and this program can only cope
> with 1.2Kb at a time.
> If people have any advice or any references that they could give me I would be eternally grateful!!
> To all those doing their Ph.D., hang on in there!!
>                            Thanks in advance,
>                            Una Fairbrother
>                            HBGU, UCL
>                            4 Stephenson Way
>                              London NW1 2HE
>                              yedwards. at uk.ac.crc
> P.S. Thats my boss' mail file so be nice!!!
> 

Is your sequence A+T rich?  (e.g. > 75%).  That can do it.  Alternatively,
do you know what the clone codes for?  There are lots of proteins that would
not be particularly healthy for an E. coli cell to express.  

Andrew Cockburn
USDA



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