Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
You should add some salt when you anneal!!! The duplex is highly negatively charged, so adding even a small amount (like 10mM NaCl) will help with charge screening, thus making the two strands less repellant to each other. Buffer is also always a good idea. At low pH and high temp you'll hydrolyze the glycosidic bonds of your purines. Also, depending on how you purified the DNA after synthesis, the white precipitate may well be other crud - protecting groups, etc. Did you desalt it at least? DNA should be very highly soluble, so your complex protocol should depend mostly on the protein's personality. If it is also highly soluble, just mix them. Finally, if you try crystallization with each possible duplex one at a time, you'll be a post-doc for a very long time. We usually try at least a half-dozen at a time (with varying ends), and it usually takes several different crystal forms to get one that diffracts decently. Good luck! Phoebe 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
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
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
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
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
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
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
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
I think that at this point you're better off looking at a professional literature. For example: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=102833 Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Sunday, June 22, 2008 1:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] query on DNA-protein complex preparation for crystallization 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
[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
Re: [ccp4bb] query on DNA-protein complex preparation for crystallization
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
