detect dsDNA (also SYBR Safe vs Ethidium Bromide)

From: zq deng
Date: 1 October 2011 04:36

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

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From: 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

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From: 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

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From: 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 crystals

Acta Crystallographica Section D

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

DOI: 10.1107/S0907444905035365

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From: 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

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From: 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

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From: 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

--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
*******************************************

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From: 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

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From: Edward A. Berry


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.

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From: 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

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From: Dima Klenchin

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

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From: Jacob Keller

Can you do the toothbrush test with SYBR Safe?

JPK

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From: 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

Tommi Kajander, Ph.D.


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From: Dima Klenchin


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