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Plasmid DNA contamination


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5 replies to this topic

#1 harry348

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Posted 14 July 2013 - 11:29 PM

Hello Guys,

I have a problem with my transfections.
I recently isolated pDNA(plasmid DNA) for transfection using Qiagen Midi prep kit and used the elute for transfections.
But after seeding the cells (adhearent cells) and transfection, i find that the cells are contaminated.
I do not know what happened.Is it due to pDNA contamination, If yes what would be the best option to purify pDNA.

PLEASE HELP ME.

thanks in advance.

Harry

#2 leelee

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Posted 14 July 2013 - 11:35 PM

What do you mean the cells are contaminated? Contaminated with what?

#3 harry348

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Posted 14 July 2013 - 11:39 PM

There is some white stuff floating in the wells.Almost covered the whole well

Edited by harry348, 14 July 2013 - 11:40 PM.


#4 harry348

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Posted 14 July 2013 - 11:41 PM

Could that be some ecoli from maxi purification steps???? sheared genomic dna?

#5 bob1

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Posted 15 July 2013 - 01:17 AM

White fluffy stuff is likely to be fungus - your best bet will be to throw out the cells and start again. To test if it is the DNA, add some of your prep to an uncontaminated well, you should see the same colonies forming. DNA is pretty much invisible in solution, so it is unlikely to be that. E. coli would look like a faint haze in the background and would have the characteristic smell.

If it is fungus, it is most likely to have come from the incubator.

As it is plasmid DNA you are working with, it is probably easiest to do a fresh prep rather than worry about the source.

#6 leelee

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Posted 15 July 2013 - 11:01 PM

What bob1 said :)




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