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DNase I digestion


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4 replies to this topic

#1 moljul

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Posted 27 April 2009 - 07:29 AM

hallo there,

i have to add a DNase I digestion step following my RNA extraction. how will i know if this digestion worked or not? agarose gel?

maybe you have some recommendations/suggestions!

#2 tea-test

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Posted 27 April 2009 - 07:51 AM

just add an RTminus control. Do duplicate reverse transcription reactions with your samples but leave out the RT enzyme (=RTminus control) in one (just add water instead). Then do PCR with both. If your primers detect gDNA and there was some gDNA left in your RNA prep you will get a signal in the RTminus control.
tea-test: The artist formerly known as Ned Land

#3 moljul

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Posted 27 April 2009 - 08:06 AM

just add an RTminus control. Do duplicate reverse transcription reactions with your samples but leave out the RT enzyme (=RTminus control) in one (just add water instead). Then do PCR with both. If your primers detect gDNA and there was some gDNA left in your RNA prep you will get a signal in the RTminus control.


right, i thought so complicated!

#4 mastermi

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Posted 27 April 2009 - 09:43 AM

You don't need to do duplicate reverse transcription without the enzyme.
Just take some of your RNA sample as template for standard PCR!

#5 AquaPlasmid

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Posted 14 May 2009 - 10:40 PM

Yes, you should take an aliquot and run the gel. You might see all your RNA's gone (over digest) or you might see a large DNA smear (under digest).




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