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Sterilize DNA preps


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5 replies to this topic

#1 dvddecarvalho

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Posted 05 July 2009 - 09:51 AM

Hi everyone,
I`m trying to transfect some cells with my DNA prep but I realized that it is contaminated.
How do you sterilized your DNA preps to transfect? Can I filter it on a 0,22um?

Thx,
DVD

#2 HomeBrew

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Posted 05 July 2009 - 12:39 PM

Ethanol precipitation, maybe? I should mention that I don't work in cell culture, so I don't know how that might effect downstream use, if at all. But EtOH ppt would take care of the contaminant, assuming the contamination is an organism. But what about the contaminating organism's DNA, of which there will surely be traces in your prep no matter how you clean it up. Will that interfere? Can you transform some bacterial cells and re-isolate your DNA, assuming your DNA is a replicon?

#3 leelee

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Posted 05 July 2009 - 06:30 PM

I would just do a new DNA prep. I think in the end it will save you time rather than running the risk of contamination.

#4 dvddecarvalho

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Posted 07 July 2009 - 05:34 AM

Thank you,
Yes, I got organisms contamination on my DNA maxiprep.
I transfect my DNA and 2 days after my culture is contaminated. I have controls showing that anything on procedure is contaminated, only DNA.
I`m not sure that just doing a new prep would resolv.
I did this prep very carefully, using qiagen collumns. What I didn`t do was to elute my DNA from collumn in a sterile enviroment. Is that necessary?

Thx,
DVD

#5 HomeBrew

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Posted 07 July 2009 - 06:38 AM

Are the kit components (buffers, columns, etc.) sterile?

If traces of the cellular components (DNA, proteins, etc.) from whatever contaminant is present won't interfere with your assay, you can ethanol precipitate the DNA -- the ethanol will kill the contaminating organism. You could also probably heat your DNA to kill the bug, provided it isn't heat tolerant and you cool your sample very slowly to allow your prep DNA to re-anneal correctly.

Personally, I would filter all buffers in the kit, and try the prep again.

#6 klinmed

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Posted 07 July 2009 - 07:00 AM

Hi everyone,
I`m trying to transfect some cells with my DNA prep but I realized that it is contaminated.
How do you sterilized your DNA preps to transfect? Can I filter it on a 0,22um?

Thx,
DVD

For tranfection I usually use DNA purified with Qiagen anion exchange columns. I run the plasmid prep exactly according to the instructions, including the isopropanol precipitation. I do not sterilize any of the solutions but, of course, use sterile plasticware.

The day before transfection I pipette twice the amount of DNA needed into a sterile microfuge tube. It is then ethanol precipitated. I then fill the microfuge tube with 70% ethanol and leave overnight at 4 oC. The next day, in a LAF bench, I remove the ethanol, dry the pellet and resuspend in a sutable volume of TE. After quantitation by A260 I use the DNA for the transfection. Never had contamination with this approach.

Hope this helps.




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