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DNA contamination in RNA extraction


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6 replies to this topic

#1 pon103

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Posted 30 March 2005 - 01:08 AM

Hi everyone,
I'd like to ask you if it is possible to overcome DNA contamination during RNA isolation. I use the TRIzol method and I always have DNA contamination.

Thanks in advance <_<

#2 kean_guy

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Posted 30 March 2005 - 01:41 AM

You can (1). do selective precipitation using lithium chloride, or
(2). use Rnase-free DNase enzyme.

Salut

#3 fred_33

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Posted 30 March 2005 - 01:56 AM

hi
i use trizol and lucky i never get contamination. Try increase the volume of trizol you use.
fred

#4 Marvilla

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Posted 30 March 2005 - 07:25 PM

Hi,
There is a modification of the guanidinium thiocianate-phenol method (trizol extraction) which reduces genomic DNA contamintion: Nucleic Acid Research, 1993m vol. 21 N 8 2019-2020. The modification involves the addition of a brief RNA selective precipitation step following the lysis of the cells with the guanidinium thiocianate solution. This is achieved by the addition of 1/3 volumen of 95% ethanol and incubation of the mixture on ice for 5 minutes. Following centrifugation for 15 minutes the supernatant is removed and the RNA pellet dissolved in 1/2 of the original volume of guanidinium thiocianate solution.

#5 methylnick

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Posted 30 March 2005 - 08:46 PM

Hi everyone,
I'd like to ask you if it is possible to overcome DNA contamination during RNA isolation. I use the TRIzol method and I always have DNA contamination.

Thanks in advance :)

<{POST_SNAPBACK}>


you can never get rid of DNA completely from an RNA prep. I have used Ambions Turbo DNA-free after Trizol isolation, it's an RNAse free DNAse that you can use to treat your preps, for the work I have done with it, DNA contamination was not a problem.

:blink:

#6 pon103

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Posted 31 March 2005 - 12:52 AM

Thanks all of you!!!!

Marvilla you're great!!! :)

#7 montie420

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Posted 31 March 2005 - 02:24 PM

While recovering the supernatant after trizol addition; do not go close to interface.




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