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Measurement of RNA concentration after Dnases treatment


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4 replies to this topic

#1 wntiong

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Posted 22 April 2010 - 08:33 PM

Hi, i did dnases treatment to all my rna samples before go to rt-pcr steps. I only stop dnases reaction after the treatment at 65c, 10 min and no further clean up was done. Just want to know, should i quantitate my rna after dnases treatment, or i can just directly use the rna for rt without measurement? I used 2 ug total rna and dnased-treated them. Can i just assume after dnases treatment, the amount of rna is the same as what i put for starting?

thanks.

#2 Curtis

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Posted 23 April 2010 - 02:44 AM

2ug is a lot and is more than enough.

if using Nanodrop I've never experienced much difference in RNA concentration before and after DNase addition.

What you can do is to use a blank that has the same amount of DNase inside, this would give you a more accurate conc.

#3 bob1

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Posted 25 April 2010 - 04:15 PM

DNases don't digest the DNA completely into bases, so there are fragments left over that are too small to amplify/worry about. However, these fragments will still be picked up by the spec.

#4 wntiong

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Posted 27 April 2010 - 08:41 PM

DNases don't digest the DNA completely into bases, so there are fragments left over that are too small to amplify/worry about. However, these fragments will still be picked up by the spec.


do you is it ok if i just proceed to reverse transcription without uv spec it? the reading is so funny after i uv spec, it gives me concentration shoot up to 3-5 ug rna, and i dun hv the nanodrop in my faculty. can i just assume i take out 10ul equivalent to 1ug rna and proceed to rt? i prepared 20ul dnased mixture with initial sample 2ug rna. thanks

#5 bluedoozer

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Posted 27 April 2010 - 09:42 PM

DNases don't digest the DNA completely into bases, so there are fragments left over that are too small to amplify/worry about. However, these fragments will still be picked up by the spec.


do you is it ok if i just proceed to reverse transcription without uv spec it? the reading is so funny after i uv spec, it gives me concentration shoot up to 3-5 ug rna, and i dun hv the nanodrop in my faculty. can i just assume i take out 10ul equivalent to 1ug rna and proceed to rt? i prepared 20ul dnased mixture with initial sample 2ug rna. thanks



well actually I would just do a column based cleanup for perfect quantification, and if this is not available just assume that your input is still the valid concentration and proceed with RT.




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