Hi Artem Evdokimov Thank you for the mail. I have synthesized DMT-on oligos in our laboratory. Deprotection was performed treating with ammonium hydroxide for 15 hours at 55C. Then, DMT-on oligo was separated from off using RPHPLC. DMT was cleaved by treating with 20% glacial acetic acid for one hour. Then, DMT-off DNA was separated from DMT, again using RPHPLC. Lyophilized DMT-off oligos were dissolved in 3 mL of Milli Q water and dialysed against 2 L of milli Q water for 4hrs by changing water 2 times.
Then complemntary oligos are concentrated around 1.0 mM and mixed them and concentrated further to 1.5 mM (duplex). 2 mM (final concentration) of Magensium chloride was added to oligos and concentrated to half of the volume. While concentrating oligos become viscos and white precipitate. however, annealing did not help to dissolve the white precipitate. I kept oligos in distilled water, without adusting pH. Please can you mail if I iginite DNA on metal spatual, eiether burns or not, what it indicates? Thanking you Rajakumara <[EMAIL PROTECTED]> wrote: > 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 > 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
