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Storing regenerated oligo DT

Fri Jul 22 15:53:48 EST 1994

>To:             methods-and-reagents at net.bio.net
>From:           acc4636 at tamsun.tamu.edu (Anthony C.)
>Subject:        Storing regenerated oligo DT
>Date sent:      22 Jul 1994 09:17:58 -0500

>I just bought two oligo Dt columns from stratagene for purifying mRNA
>from the looks of it if I ever lose these two columns I know how to
>make them on my own :)  But for now, the company only guarantees each
>column for 1 use however they said the columns can be regenerated 
>according to maniatis et al.  Now I looked at it and I understand
>how it can be regenerated, but the instructions leave a couple of questions.
>1) What buffer does the column need to be stored in (1X column loading

We always store our Gibco Push oligo-dT columns moist in the freezer.

>2) how many times can the column practically be regenerated

We have used the push columns 6-10 times.  We strip with a couple rinses 
with 100 mM NaOH and then neutralize with 3-4 rinses with TNE.

>3) does anyone have any experience with the stratagene poly A quik
>mRNA isolation kit? good or bad etc.. any tips?
>4) Im loading 100 ug or so of my total RNA to make a cDNA library
>my cells are spleen cells stimulated with LPS so there should be a 
>large proportion of mRNA, but whats the best way to quantitate the 
>the amount of undegraded mRNA before I do the expensive cDNA
>synthesis procedure.

We quantitate by UV spectrophotometry using microcuvettes.  We also 
usually run 1-2 ug on a standard 1.2% agarose gel stained with EtBr.  
Since not all of the rRNA is removed you still should see these 
bands.  If you don't then you probably have had significant degradation.

Doug Rhoads                  || Dept. of Biological Sciences
drhoads at mercury.uark.edu     || 601 Science Engineering
drhoads at uafsysb.uark.edu     || University of Arkansas
501-575-3251                 || Fayetteville, AR 72701

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