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Measurement of RNA concentration after Dnases treatment - (Apr/22/2010 )

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.

-wntiong-

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.

-Curtis-

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.

-bob1-

bob1 on Apr 26 2010, 08:15 AM said:

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

-wntiong-

wntiong on Apr 27 2010, 09:41 PM said:

bob1 on Apr 26 2010, 08:15 AM said:

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.

-bluedoozer-