Help with cDNA
William W.-G. Jia
weiguo at ecc.ubc.ca
Wed Feb 23 22:02:56 EST 1994
The only thing I can help you with (I hope) is the first question.
In article <199402221805.KAA13779 at net.bio.net> , MUNGO at UVVM.UVIC.CA
>1) I wish to isolate polyA+ RNA from a large sample of small (but not
>precious) tissue samples for RTPCR. I would like a method whereby I
>oligo dT matrix to select straight out of the guanidinium lysis buffer. I
>would also like to wash and elute from the matrix using a small spin
>(similar to InVitrogens micro fast track kit). Does anyone have
>or better yet detailed protocols. Any suggestions on the oligo dT
>I have tried a number of oligo dT cellulose matrices in the spin columns
>they tend to plug (I have removed the fines from the matrix).
Why don't you try oligo dT Dynabeads (magnetic beads) available from
Dynal. I think they have a kit out in which you can just homogenize the
tissue and mix in Dynabeads to isolate poly A mRNA. This way you don't
even have to use spin columns to elute the mRNA (you can elute directly
from the beads). We usually isolate total RNA first (in order to check
the quality of the RNA) and then we use Dynabeads to isolate mRNA. The
mRNA we get is very clean and we have had good success with the beads.
Another good thing about the beads is that they are somewhat reusable
(after eluting the mRNA, you can actually reuse the beads to elute more
mRNA). However, please note that we have only reused the beads on the
same day and on the same type of sample. Hope this helps.
(I don't work for any company)
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