oligodt-cellulose

[Szabadkai Gyorgy]

I have some question about preparing mRNA on oligodT-cellulose. 
We prepared polyA+ RNA from total RNA prepared from rat skeletal 
muscle with the the guanidiumHCl/phenol extraction method. For 
binding we used a 0.5M LiCl/0.1%SDS/1 mM EDTA/10mM Tris/pH7.5 
solution, for middle wash we used the same but 1.5 M LiCl and we 
eluted tha polyA+ fraction with TE, then precipitated with 0.3M 
NaAc/70% ethanol. All the solutions were treated with DEPC as 
recommended (except for Tris, which was filtrated through steril .45 
filter, and autoclaved). Then we electrophoresed and blotted the the 
total RNA, the run-through (polyA-), the washing, and the polyA+ 
fractions adn hybridized first with GAPDH probe (GADH mRNA - 1.4 kB) 
then with a calcium channel probe (SKa1 mRNA size 6.5 kB) to check 
the integrity and enrichment of mRNA. THe yield of mRNA was 20 ug 
from 1.1 mg total RNA on a 0.5 mL Invitrogen oligodt cellulose matrix. 
The small message enrichment was OK, about 30 times. But the larger 
message somehow disappeared. Most of it appeared in the middle wash 
fraction and was significantly degraded. It is surprising because the 
GAPDH message seemed to be intact.
We would like to use our mRNA to microinject in oocytes, but if it 
does not not contain the mRNAs of larger size, it is not appropriate 
for us.
Some months ago we tried NEB oligodt-cellulose and the 
result was similar.
If you have some tips how to solve this problem, please contact me.



Thanks in advance,

Gyorgy Szabadkai





 the adress of 
your closest local distributor to Hungary
> >and a price list of your products including total and mRNA isolation
> >kits, if possible by email: szabadkai at puskin.sote.hu, or by fax to :
> >Dept of Physiology, Semmelweis Univ. of Medicine, 36-1-266-6504
> >
> >Thanks in advance,
> >
> >
> >Szabadkai Gyorgy
> 
> Invitrogen Technical Services
> Europe
> 
> 
> Visit us at the Web:
> http://www.invitrogen.com
> 
> 
> 
>