RNA transferring

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:
> Hi,netters:
>    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 
8:370-371.

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
===========================================================================
+  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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