Unfortunately for you, the optimal length for binding is not necessarily
the optimal length for crystallization.
On 10/27/15 11:15, Antonio Ariza wrote:
> Hi all,
>
> I'm trying to co-crystallise an enzyme with a ssDNA substrate. The ssDNA I used is 9 nucleotides long and as it is fairly short we had it synthesised by EUROFINS as a cloning primer using a synthesis scale of 1 micromol and HPSF as the purification method.
>
> We have established the optimal ssDNA sequence for binding and the enzyme clearly binds the DNA in gel-shift assays ... but I get no crystals using 11 different 96-well screens at both 20 and 4 degrees C. Obviously there a myriad of parameters I could change for my next screens, but I'm going to start by trying different lengths of ssDNA next, both shorter and longer.
>
> I'm wondering if using primers as a source of ssDNA is ok and if they are pure enough. What sort of purity and DNA synthesis company has worked for you?
>
> Cheers,
>
> Tony
>
> ------------------------------------------------------
>
> Dr. Antonio Ariza
> University of Oxford
> Sir William Dunn School of Pathology
> South Parks Road
> Oxford
> OX1 3RE
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All Things Serve the Beam
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David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
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