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
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Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
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