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
