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Problem detecting RNA

Dhundy Raj Bastola dbastola at CHRISTA.UNH.EDU
Sun Jul 24 15:43:02 EST 1994


Hi Netters (especially RNA people):=20

I have been using Genius kit to detect nucleic acid from my trangenic
carrot cells, Southern works beautifully.  I did have a lot of problems
isolating high molecular weight DNA until this last method where I do not
use phonol .=20

Since most of the other methods use phenol quite often i modified the same
DNA isolation protocol so that I will have to use phenol only once. The
r=E2esult is satisfactory in the sense that yield is good and denaturing ge=
l
do not show degradation. However, I am having problems with Northren. When
I do a dot blot with the sample, there is a faint signal with 10x the
amount of total RNA but no signal with the regular 20 ug. I wonder if i am
=E2loosing a lotof my mRNA during extraction because of the introduction of
Phenol treatment following 15 min incubation with DNAse or the detection
method is not sensitive enough to detect smally quantities. Since I have a
positive signal of expected size in the western I do not believe there is
no mRNA. I would highly appreciate if any one Could suggest a better non
radioactive method or help me point out the steps I probably overlooked?
=20
Thank you in advance.=20

Kiran




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