Hi, Water is not very nice a medium for a polyanion such as DNA when concentrated. Better use a basic buffer, even 10 mM Tris pH 8.0-8.5 would do. Another important thing is, as you have already partly tried, ionic strength but also the nature of the ions. You need monovalent cations as well. I would dissolve DNA in say 10 mM Tris pH 8.0, 100 mM NaCl and 5 mM MgCl2.
Having said that, it's a bit strange that you get such precipitate with a not so long DNA... I suppose you have tried to make sure it's DNA and not some chemical left from its synthesis. You may want to try and resuspend it in the buffer I mentioned above and then leave it overnight in the fridge at a concentration of say <=100 uM of duplex (so ~<=1mg/ml) Next day you can try to repeat the annealing, leaving at least 5-10 min at 90-95 ºC and then slowly decrease temperature (0.5 ºC/min should be fine) to 4 ºC. The way you prepare a DNA/protein complex depends on many factors, including the affinity of their interaction. If there is a strong interaction, I would mix them at a lower concentration at whatever relevant stoichiometry ( possibly trying as well slightly changed stoichiometries to allow for your errors in determining concentrations ) and then concentrate the whole complex. I hope this helps. Miguel 2008/6/21 E rajakumar <[EMAIL PROTECTED]>: > Dear All > Sorry for non-crystallography question. I have > synthesized two complementary strands of 16 bases in > length for making duplex DNA and co-crystallization > with DNA binding protein. I have mixed two > complementary strands of 1:1 molar ratio (0.5 mM) in > water and concentrated to 1.5 mM (Duplex), while > concentrating solution becomes viscous and turned to > white precipitate. However, adding 2 mM Magnesium > chloride followed by annealing (heating at 90C for 10 > minutes and followed by cooling to room temperature) > did not help to dissolve the white precipitate. > > Please can you give me suggestions on following > queries? > > 1.How do I dissolve white precipitate? Is increasing > divalent cation or keeping duplex in particular pH > could help in dissolving the precipitate? > 2.How do I prepare DNA-protein complex? I mean, can I > mix diluted DNA and protein in 1:1 molar ratio and > concentrate further? > Any guidance in this regard will be appreciated. > > Thank you in Advance > > Rajakumara > > > > E. Rajakumara > Postdoctoral Fellow > Strcutural Biology Program > Memorial Sloan-Kettering Cancer Center > New York-10021 > NY > 001 212 639 7986 (Lab) > 001 917 674 6266 (Mobile) > > > Send instant messages to your online friends http://uk.messenger.yahoo.com > -- http://www.pangea.org/mol/spip.php?rubrique2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Je suis de la mauvaise herbe, Braves gens, braves gens, Je pousse en liberté Dans les jardins mal fréquentés! Georges Brassens
