Re: [ccp4bb] detect dsDNA

[Dima Klenchin]

>> There exists a less toxic chemical than EtBr to stain DNA: SYBR safe
>> DNA stain (a fluorescence dye sold by a certain vendor).
>
> SYBR Safe is about 10X less sensitive though.

Can you do the toothbrush test with SYBR Safe?


I wouldn't do that. As it is considerably more hydrophobic, I'd expect it 
to have more interactions with the body than EthBr. If anything, I expect 
SYBR Safe to be a lot less safe than EthBr.


- Dima

Re: [ccp4bb] detect dsDNA

[Tommi Kajander]

i wouldn't recoommend that. here is the info from somebody forwarded  
from our genetics department with regards to safety of that while  
back


"Sybr is just as toxic/poisonous/harmful as ethidium bromide, only  
far more expensive. There have been very few tests concerning it's  
use up until now and therefore it should be treated with even more  
caution than ethidium bromide. Waste must be separated and processed  
by the company  and use of Sybr in the lab must involve safety and  
care levels at least as stringent as with Ethidium bromide."


(not sure thow if it was SYBR-"safe"...  safe must mean safe, right...  
in particular with chemical companies...)


Feel free to handle your gels as sloppy as you like (or eat them or  
whatever), but mind the other people's health in the lab.  There are  
people who actually have to work with this stuff for decades. I would

advice following the safety instructions.

Tommi



On Oct 2, 2011, at 9:10 PM, Jacob Keller wrote:


There exists a less toxic chemical than EtBr to stain DNA: SYBR safe
DNA stain (a fluorescence dye sold by a certain vendor).


SYBR Safe is about 10X less sensitive though.



Can you do the toothbrush test with SYBR Safe?

JPK

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***



Tommi Kajander, Ph.D.
Junior Group Leader
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1
(P.O. Box 65)
00014 Helsinki
Finland
p. +358-9-19158903
tommi.kajan...@helsinki.fi

Re: [ccp4bb] detect dsDNA

[Jacob Keller]

>> There exists a less toxic chemical than EtBr to stain DNA: SYBR safe
>> DNA stain (a fluorescence dye sold by a certain vendor).
>
> SYBR Safe is about 10X less sensitive though.


Can you do the toothbrush test with SYBR Safe?

JPK

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] detect dsDNA

[Dima Klenchin]

There exists a less toxic chemical than EtBr to stain DNA: SYBR safe
DNA stain (a fluorescence dye sold by a certain vendor).


SYBR Safe is about 10X less sensitive though.

I suspect that not many chemicals in the lab are less toxic/mutagenic than 
EthBr. The classic Ames test shows that 5 ug of EthBr results in 1012 
revertants. In comparison, condensate from a smoke of a a single cigarette, 
in comparison, gives almost 20X more revertants (18200, Table 1 in: PNAS, 
1975, 72(12):5135. Detection of carcinogens as mutagens in the 
Salmonella/microsome test: Assay of 300 chemicals").


Assuming linear relationship, it means that one has to eat about ten mini 
agarose gels (20 ml at 0.5 ug/ml each) to get approximately the same 
mutagenic effect as smoking a single cigarette. :-)


Additional reason why EthBr is probably completely harmless to humans at 
concentrations that we use it is the fact that it is so hydrophilic that it 
cannot pass plasma and instead is very quickly cleared from the bloodstream 
by kidneys.


- Dima

Re: [ccp4bb] detect dsDNA

[Florian Schmitzberger]

There exists a less toxic chemical than EtBr to stain DNA: SYBR safe  
DNA stain (a fluorescence dye sold by a certain vendor). Another  
benefit is to be able to use blue light, reducing UV/VIS light  
exposure when handling gels.


Florian

On Oct 2, 2011, at 11:49 AM, Edward A. Berry wrote:


Jacob Keller wrote:

I actually looked at an EtBr MSDS a while ago, and was shocked at how
benign it was. I also heard from someone that they used to feed it to
Argentinian cows routinely a few years back...



Wikipedia says it was used as a trypanosomacidal - It's being
discontinued not because of toxicity to beast or man, but because
of insufficient toxicity to trypanosomes- the little buggers
are developing resistance. Of course resistance would develop
earlier if EtBr is mutagenic. Maybe they overexpress
DNA repair enzymes.

Re: [ccp4bb] detect dsDNA

[Edward A. Berry]

Jacob Keller wrote:

I actually looked at an EtBr MSDS a while ago, and was shocked at how
benign it was. I also heard from someone that they used to feed it to
Argentinian cows routinely a few years back...



Wikipedia says it was used as a trypanosomacidal - It's being
discontinued not because of toxicity to beast or man, but because
of insufficient toxicity to trypanosomes- the little buggers
are developing resistance. Of course resistance would develop
earlier if EtBr is mutagenic. Maybe they overexpress
DNA repair enzymes.

Re: [ccp4bb] detect dsDNA

[Poul Nissen]

We chewed pills with EtBr as kids in school to see if we brushed our teeth well 
- red colour on the edges, bad boy

Poul



On 01/10/2011, at 19.12, Jacob Keller  wrote:

> I actually looked at an EtBr MSDS a while ago, and was shocked at how
> benign it was. I also heard from someone that they used to feed it to
> Argentinian cows routinely a few years back...
> 
> JPK
> 
> On Sat, Oct 1, 2011 at 1:56 AM, James Stroud  wrote:
>> If you can reproduce the crystals and have the material
>> 
>> 1. Harvest several large crystals.
>> 2. Make several transfers to fresh mother liquor to wash.
>> 3. Dissolve in DNA loading dye without SDS
>> 4. Run on a native gel (e.g. 6% polyacrylamide, 0.5XTBE, etc.).
>> 5. Include positive control lanes for protein, DNA, and complex.
>> 6. Stain with ETBr. Take a picture.
>> 7. Wash out the ethidium in accordance with state, local, federal, UN, laws, 
>> filling out the proper documentation ad nauseum. Take a safety class just to 
>> be sure you didn't miss something. Hug a bureaucrat.
>> 8. Stain with coomassie. Take a picture.
>> 
>> That should tell you more than you need to know.
>> 
>> James
>> 
>> On Sep 30, 2011, at 9:36 PM, zq deng wrote:
>> 
>>> Hi all,
>>> .
>>> recently,I got a crystal of protein-DNA crystal.i used silver stainto prove 
>>> that it is a protein crystal.Does anyone have method to detect if there is 
>>> DNA in the crystal.
>>> any suggestion will be appreciated.
>>> 
>>> Regards,
>>> deng
>> 
> 
> 
> 
> -- 
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***

Re: [ccp4bb] detect dsDNA

[Jacob Keller]

I actually looked at an EtBr MSDS a while ago, and was shocked at how
benign it was. I also heard from someone that they used to feed it to
Argentinian cows routinely a few years back...

JPK

On Sat, Oct 1, 2011 at 1:56 AM, James Stroud  wrote:
> If you can reproduce the crystals and have the material
>
> 1. Harvest several large crystals.
> 2. Make several transfers to fresh mother liquor to wash.
> 3. Dissolve in DNA loading dye without SDS
> 4. Run on a native gel (e.g. 6% polyacrylamide, 0.5XTBE, etc.).
> 5. Include positive control lanes for protein, DNA, and complex.
> 6. Stain with ETBr. Take a picture.
> 7. Wash out the ethidium in accordance with state, local, federal, UN, laws, 
> filling out the proper documentation ad nauseum. Take a safety class just to 
> be sure you didn't miss something. Hug a bureaucrat.
> 8. Stain with coomassie. Take a picture.
>
> That should tell you more than you need to know.
>
> James
>
> On Sep 30, 2011, at 9:36 PM, zq deng wrote:
>
>> Hi all,
>> .
>> recently,I got a crystal of protein-DNA crystal.i used silver stainto prove 
>> that it is a protein crystal.Does anyone have method to detect if there is 
>> DNA in the crystal.
>> any suggestion will be appreciated.
>>
>> Regards,
>> deng
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] detect dsDNA

[Phoebe Rice]

Some things that are a good sign for having DNA as well as protein in your 
crystal:

1) You get different (or no) crystals if you add or substract a base or two 
from the DNA ends.  Of course, we did get fooled by this logic once when one 
particular oligo seemed to be contaminated with an easily crystallizable 
mystery salt.

2) Unusually intense spots at ~3.4A, usually in the best direction of 
diffraction (due to the spacing between bp).  If the DNA is a reasonable 
fraction of your scattering mass, it may be quite noticeable with hklview.

3) Anisotropic diffraction, often to far lower resolution than you'd hoped for.

Also note that fragile hexagonal crystals might be DNA-only.  Especially if the 
person setting up the trays tells you they were optimized by increasing the 
DNA:protein ratio ;-).

  Good luck!
Phoebe

=
Phoebe A. Rice
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
http://www.rsc.org/shop/books/2008/9780854042722.asp


 Original message 
>Date: Sat, 1 Oct 2011 11:36:59 +0800
>From: CCP4 bulletin board  (on behalf of zq deng 
>)
>Subject: [ccp4bb] detect dsDNA  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Hi all,
>   .
>   recently,I got a crystal of protein-DNA crystal.i
>   used silver stainto prove that it is a protein
>   crystal.Does anyone have method to detect if there
>   is DNA in the crystal.
>   any suggestion will be appreciated.
>    
>   Regards, 
>   deng

Re: [ccp4bb] detect dsDNA

[Kay Perry]

If your DNA is small enough, you can run a control dsDNA lane on the gel. dsDNA 
will show up on silver stain but the oligomer has to be small enough to enter 
the gel. 

Kay

On Sep 30, 2011, at 10:37 PM, zq deng  wrote:

> Hi all,
> .
> recently,I got a crystal of protein-DNA crystal.i used silver stainto prove 
> that it is a protein crystal.Does anyone have method to detect if there is 
> DNA in the crystal.
> any suggestion will be appreciated.
>  
> Regards, 
> deng

Re: [ccp4bb] detect dsDNA

[Juha Vahokoski]

Also this method might be useful if crystals tolerate the treatment.

Regards,
Juha

Fluorescence detection of nucleic acids and proteins in multi-component
crystalsActa
Crystallographica Section D

Volume 62, Issue 2, February 2006, Pages: 146–150, Hubert Kettenberger and
Patrick Cramer

DOI: 10.1107/S0907444905035365 


On 1 October 2011 06:36, zq deng  wrote:

> Hi all,
> .
> recently,I got a crystal of protein-DNA crystal.i used silver stainto prove
> that it is a protein crystal.Does anyone have method to detect if there is
> DNA in the crystal.
> any suggestion will be appreciated.
>
> Regards,
> deng
>

Re: [ccp4bb] detect dsDNA

[James Stroud]

If you can reproduce the crystals and have the material

1. Harvest several large crystals.
2. Make several transfers to fresh mother liquor to wash.
3. Dissolve in DNA loading dye without SDS
4. Run on a native gel (e.g. 6% polyacrylamide, 0.5XTBE, etc.).
5. Include positive control lanes for protein, DNA, and complex.
6. Stain with ETBr. Take a picture.
7. Wash out the ethidium in accordance with state, local, federal, UN, laws, 
filling out the proper documentation ad nauseum. Take a safety class just to be 
sure you didn't miss something. Hug a bureaucrat.
8. Stain with coomassie. Take a picture.

That should tell you more than you need to know.

James

On Sep 30, 2011, at 9:36 PM, zq deng wrote:

> Hi all,
> .
> recently,I got a crystal of protein-DNA crystal.i used silver stainto prove 
> that it is a protein crystal.Does anyone have method to detect if there is 
> DNA in the crystal.
> any suggestion will be appreciated.
>  
> Regards, 
> deng

Re: [ccp4bb] detect dsDNA

[Dunten, Pete W.]

Try the program DIBER to evaluate the likelihood your crystal contained ordered 
nucleic acid in addition to protein.  See Acta Cryst. (2010). D66, 643-653 
“DIBER: protein, DNA, or both?” by G. Chojnowski & M. Bochtler.

Good luck, Pete

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of zq deng 
[dengzq1...@gmail.com]
Sent: Friday, September 30, 2011 8:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] detect dsDNA

Hi all,
.
recently,I got a crystal of protein-DNA crystal.i used silver stainto prove 
that it is a protein crystal.Does anyone have method to detect if there is DNA 
in the crystal.
any suggestion will be appreciated.

Regards,
deng

[ccp4bb] detect dsDNA

[zq deng]

Hi all,
.
recently,I got a crystal of protein-DNA crystal.i used silver stainto prove
that it is a protein crystal.Does anyone have method to detect if there is
DNA in the crystal.
any suggestion will be appreciated.

Regards,
deng