Isolation of mRNA for cDNA probe?

Carl Simard c.simard at sympatico.ca
Tue Oct 20 17:18:15 EST 1998


I have purified polyA to make cDNA with the Pharmacia Kit (prepacked
column with all the buffer). Everything goes rapidly and without problem
and the cDNA obtained was very good. There is no need to digest with DNAse
unless it is absolutely essential to have a DNAse free preparation.

Wish it helps!

Carl Simard
c.simard at sympatico.ca
Victor Levenson wrote:

> Hello, nettters:
>
> I want to use cDNA form my cells as a probe for differential
> screening. Since so far I was pretty happy  with total RNA on
> Northerns (Trizol isolation), I am somewhat apprehensive about mRNA
> stuff. The info that I pulled from the Web indicates that the major
> problem for cDNA probes is the presence of trace amounts of genomic
> DNA. One of the kits (Clontech's Atlas Pure RNA, no affiliation, etc)
> requires DNase digestion of total RNA BEFORE polyA+ isolation and that
> seems a bit excessive.
>  Hence the question(s): what is your experience with cDNA used as a
> probe (especially for differential screening), what procedure do you
> use to eliminate genomic DNA, is Trizol followed by polyA+ isolation
> sufficient in your opinion, and - if you are using a kit - which one
> would you recommend based on price/performance?
>
> Thanks for your input!
>
> Victor
>
> Victor Levenson MD/PhD
> Res Asst Prof
> UIC, Dept of Genetics M/C 669
> 900 S Ashland Ave                   The one who wants finds HOW
> Chicago IL 60607                    The one who doesn't finds WHY
> levenson at uic.edu
> 312-413-3887
> 312-413-3128 FAX




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