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
>
>
>
>
>
>
>
> <[log in to unmask]> 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:[log in to unmask]] On Behalf Of E
>> rajakumar
>> Sent: Saturday, June 21, 2008 5:48 PM
>> To: [log in to unmask]
>> 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