Hi, How did you synthesize the DNA? I assume external vendor (so few people make their own these days)? How was the DNA purified? Sometimes if only a 'desalting' step is used there may be 'other chemicals' in the mix. Also, what pH was your DNA at, and in what buffer (if any)? If your DNA degraded you may have Pi in solution, which forms insoluble precipitates with many counterions.
So, first of all I would check your white precipitate - does it dissolve in anything at all? If it does dissolve, what pH does it have? Does it run on an agarose gel? When you ignite a speck of it on a clean metal spatula - does it burn or does it just sit there (and what color does it become). Normally you can prepare DNA-protein complexes in a variety of ways, including direct addition, concentration, counterdialysis, etc. Regards, Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Saturday, June 21, 2008 5:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] query on DNA-protein complex preparation for crystallization 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. Sorry, foregot to mention that any references in this regards will be great help. 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
