Hi Thank you for the mail. It seems your correct. A(260 nm)/A(280) of one oligo is around 1.0 and peak is around 272. Other oligo's(260 nm)/A(280) is around 1.5. Can I know what is the absorbance peak of base protecting N-benzoyl group. Is it possible to do deprotection of base after mixing complementary strands? Can you suggest me what is the volume of ammonium hydroxide will be used for 1uM oligo of 16 bases in length and how much time heating shoul be done? thanking you rajakumara
--- William Scott <[EMAIL PROTECTED]> wrote: > Check the purity of the DNA in solution: > > A(260 nm)/A(280) = 1.8 for fully deprotected DNA, > and you should see a > nice clean simple curve with a peak very close to > 260 nm. > > Check it on a denaturing gel. Smearing indicates > incomplete > deprotection. This is usually the cause of > solubility problems. > > Sometimes resuspending in a strong cationic buffer > (say 100 mM Tris pH > 8.5) might be required. For crystallization it is > probably best to > have Na+ or K+ as a counterion, rather than Mg++. > So you need to > dialize against a high concentration of monovalent > salt first, not > just deionized water. > > > On Jun 22, 2008, at 9:10 AM, E rajakumar wrote: > > > 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 > 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
