David L. Haviland, Ph.D.
HAVILAND at KIDS.WUSTL.EDU
Fri Oct 6 13:38:20 EST 1995
In <DFxqtt.9Gw at riker.neoucom.edu> aq at uhura.neoucom.edu writes:
> The inefficient RNA transfer from agarose gel to positively charged
> nylon membrane bothers me a lot. I ran the RNA samples in
> formaldehyde denaturing gel, rinsed the gel in Rnase-free water 3
> times, and rinsed the gel in transfer buffer for 45 min. transferred
> without air bubbles. overnight. Only 70% of the RNA samples were
> transferred. I tried downward, upward transfer and electric blotting
> apparatus, tried agarose from different manufactures. Still the
> transfer didnot work efficiently, specially, the 28s rRNA. The ratio
> of 260/280 of the RNA samples is around 1.7 to 2.1. Can anybody
> give me opinions?
Sure. I'd like to make a recommendation if I may. I too had my share of
less than perfect RNA transfers using traditional methods. Then I tried
this one: "Simplified northern blot hybridization using 5% sodium dodecyl
sulfate" by Virca, Northemann, Shiels, Widera, Broome. BioTechniques
I don't want to beat my own drum, however, I have had *unqualified* and
*unequalled* success with this method. I use a small amount of EtBr (3 ul
of a 10mg/ml stock for 100 ml 3% formaldehyde gel) and can visualise the
bands just fine. With the transilluminator, after an overnight transfer in
NaPO4 buffer per the method, I have NEVER seen any residual RNA in the
agarose gel. All is usually seen on the Hybond-N+. So I would guesstimate
a 95+% transfer. I've never had a reason to quantitiate the remaining RNA.
I've never looked back at the traditional 20XSSC transfer protocol and I've
not yet tried this method with any sort of transblotter. All I use are the
Altec blotting stones.
I hope this helps.
+ David L. Haviland, Ph.D. Internet:"haviland at kids.wustl.edu" +
+ Washington Univ. School of Med. +
+ Dept. of Peds./Pulm. Box 8116 +
+ 1 Children's Place +
+ St. Louis, MO 63110 FAX: 314-454-2476 +
+ (314) 454-6076 +
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