>1. Do people routinely try different lengths of DNA? Yes, its usually the most important variable because the ends like to stack against each other to form a pseudo-continuous helix.
>2. Do you start with blunt or sticky ends? Both. Plan on a dozen or so different duplexes to start (you can usually mix-n-match top & bottom strands to get more variety at the ends). >3. Would purification of the resultant complex by gel filtration be >a good idea as the interaction is so tight? Probably wouldn't hurt, but probably not necessary. You can screen a lot more DNAs if you start out with mix-n-pray. >4. Which screens would you try first? PEG/ion AND hand-made PEG vs. salt, +/- Mg++, with concentrations that suit our complexes' personalities. >5. Where do you order the DNA from as there is a large difference >in price depending on supplier. What scale do you go for and what >purification? We've had good luck with IDT. For screening, even for lengths in the low 30s, we just use the unpurified stuff. Its much faster and cheaper that way! Then try purifying the oligos that give you hits. Sometimes it matters, sometimes it doesn't. >6. We expect 1:1 binding. What ratios of DNA to protein are >generally used (bearing in mind the inaccuracies of protein estimation)? Usually a bit of extra DNA. > >7. Any other useful tips? Protein-DNA cocrystals are often (at least in my lab) fragile and a pain to freeze. Do at least some of your screens with enough glycerol around to act as a cryoprotectant, so that you won't have to do extra crack-inducing manipulations later. > >Thanks in advance for the suggestions and advice > >Clare > >Dr. Clare E. M. Stevenson >John Innes Centre, >Department Biological Chemistry >Colney Lane >Norwich >Norfolk >NR4 7UH Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
