northern blot

Ed Rybicki ED at micro.uct.ac.za
Thu Dec 3 14:38:08 EST 1992


Here, for all the requests I have had personally, and all that have come up
on the net generally, is THE DEFINITIVE Northern Blot With Digoxygenin
Technique - by Andy Hackland of the Dept of Microbiology, UCT.  Endorsed
by Maud Purves, PhD, and used with success by several here.  Enjoy!


------- Forwarded Message Follows -------

To:            micro/ed
From:          MICRO/ANDY
Date:          3 Dec 92 13:44:20 SAST-2
Subject:       northern blot

Northern analysis using non-radioactive RNA transcript
probes labeled in vitro with the hapten digoxigenin (DIG)

Procedure for the detecton of membrane-bound RNA with
Lumigen-PPDTM substrate

Total plant RNA was isolated using the rapid small scale
procedure of Verwoerd et al (1989).  Approximately 10ug of
RNA was separated in a 1% agarose gel under non-denaturing
conditions, that is, no formaldehyde was present in the gel.
The RNA was then transferred to a nylon membrane (Hybond N+,
Amersham) via a dry alkali blotting procedure.

RNA alkali blotting procedure:

1.  Place gel on 2-3 presoaked Whatman 3MM filters, wetted
with 0.05M NaOH.  Use 0.05M NaOH for RNA.

2.  Cut a sheet of Hybond N+ to the exact size of the gel
and place on top of the gel.

3.  Place another 2-3 presoaked filters, cut to size on, top
of the membrane.

4.  Place a stack of absorbent paper towel on top of the 3MM
paper, and put a 1kg weight on top.  Allow the transfer to
proceed for 2-3 hours.

5.  THERE IS NO NEED TO FIX THE RNA AFTER ALKALI BLOTTING.

6.  Remove the membrane and place RNA side up on presoaked
whatman 3MM, wetted with 0.05M NaOH, and leave for 5 min.

7.  Rinse the membrane in 2x SSPE with gentle agitation.

NOTE: All these procedures were carried out under RNase
sterile conditions, as the probe to be used was a RNA
transcript.

Prehybridization was carried out using the protocol outlined
by Boehringer Mannheim Biochemica, for DIG nucleic acid
detection.  Hybridization buffer is made as follows, 50%
formamide; 5x SSPE; blocking reagent (Boerhinger), 2%(w/v)
(added from a 10% RNase sterile blocking stock solution); N-
lauroylsarcosine, 0.1% (w/v); SDS, 0.02%(w/v).  This buffer
can be stored frozen at -20oC.  The membrane was
prehybridized in a sealed plastic bag with 20 ml
hybridization buffer per 100cm2 of membrane at 68oC for 1 h.
The solution was replaced with 2.5 ml of hybridization
buffer per 100 cm2 of membrane.  100 ng of heat denatured
probe was added and the membrane incubated at 68oC
overnight.  The following steps were not carried out under
RNase sterile conditions.  The membrane was then washed 2 x
5 min at room temperature with 2 x SSPE; SDS, 0.1% (w/v),
and 2 x 15 min at 68oC with 0.1 x SSPE; SDS, 0.1% (w/v).

Immunological detection of the DIG hapten was carried out
exactly as described by Boehringer Mannheim.

References:

Holtke, H-J, and Kessler, C. 1990.  Non-radioactive labeling
of RNA transcripts in vitro with the hapten digoxygenin
(DIG); hybridization and ELISA-based detection.
NAR.18(19):5843-5851

Verwoerd, T.C., Dekker, B.M.M., and Hoekema, A. 1989. A
small-scale procedure for the rapid isolation of plant RNAs.
NAR.17(6): 2362
  ____________________________________________________________________
 | Ed Rybicki, PhD             |    "Now you've got the hang of it    |
 | (ed at micro.uct.ac.za)        | There's nothing you can't do with it |
 | Dept Microbiology           |        If you're very into it        |
 | University of Cape Town     |         You can't go wrong...."      |
 | Private Bag, Rondebosch     |                                      |
 | 7700, South Africa          |               -Mad John              |
 | fax: 27-21-650 4023         | (Ogden's Nut Gone Flake, Small Faces)|
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