downward northern (no html)

Bradley Turner turner at sprcore.caregroup.harvard.edu
Fri Mar 10 14:07:47 EST 2000


1: Biotechniques 1998 Sep;25(3):420-3, 425-6

Highly sensitive northern hybridization using a rapid protocol for
downward
alkaline blotting of RNA.

Ingelbrecht IL, Mandelbaum CI, Mirkov TE

Texas A&M University System Agricultural Experiment Station, Weslaco,
USA.

A simple and fast RNA gel blot procedure is described that uses 50 mM
NaOH to
simultaneously transfer and fix RNA to a positively charged nylon
membrane. The
RNA is transferred in a downward direction, and transfer is routinely
completed
within 2.5 h. The resulting blots give increased sensitivity over
existing
methods without affecting RNA integrity and can be used in both
radioactive and
nonradioactive detection procedures.

Publication Types:
Technical report

PMID: 9762439, UI: 98435187



2: Anal Biochem 1992 Feb 14;201(1):134-9

One-hour downward alkaline capillary transfer for blotting of DNA and
RNA.

Chomczynski P

Division of Endocrinology, College of Medicine, University of
Cincinnati, Ohio
45267.

The downward alkaline capillary transfer of DNA and RNA from agarose gel
to a
hybridization membrane was performed using a transfer solution
containing 3 M
NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were
completely
eluted from the agarose gel and bound to a hybridization membrane within
1 h. On
the basis of this new method of transfer a blotting protocol, downward
alkaline
blotting, was elaborated. It provides a fast and efficient alternative
to
commonly used Southern and Northern blotting protocols. The downward
alkaline
blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively,
and can
be used with both plastic and nitrocellulose membranes. In addition, the
downward alkaline blotting protocol allows for a hybridization
efficiency of DNA
and RNA higher than that of the standard blotting protocols performed at
neutral
pH.

PMID: 1621951, UI: 92321447



3: Biotechniques 1993 Aug;15(2):260-2

A rapid optimized protocol for downward alkaline Southern blotting of
DNA.

Koetsier PA, Schorr J, Doerfler W

Institute for Genetics, University of Cologne, FRG.

A simple and efficient 2.5-h Southern blotting procedure is described
that uses
0.4 M NaOH to transfer DNA in a downward direction. The resulting blots
give
signals that are both sharper and 30% stronger than those obtained by
conventional upward-transferred blots.

Publication Types:
Technical report

PMID: 8373591, UI: 93384831



4: Am Biotechnol Lab 1993 Nov;11(12):38

Rapid, efficient downward blotting of DNA and RNA.

La Roche J

Schleicher & Schuell, Inc., Keene, NH 03431.

PMID: 7694596, UI: 94059496



5: Biotechniques 1994 Jan;16(1):58-9

Improved downward capillary transfer for blotting of DNA and RNA.

Ming YZ, Di X, Gomez-Sanchez EP, Gomez-Sanchez CE

Research Service, Harry S. Truman Veterans Hospital, Columbia, MO 65201.

PMID: 8136141, UI: 94183561



6: Anal Biochem 1990 Nov 15;191(1):187-91

A procedure for DNA and RNA transfer to membrane filters avoiding
weight-induced
gel flattening.

Lichtenstein AV, Moiseev VL, Zaboikin MM

Laboratory of Tumor Biochemistry, All-Union Cancer Research Center,
Moscow,
USSR.

We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA
from
agarose gels to nitrocellulose or nylon membrane filters. It is
characterized by
nearly complete elimination of mechanical action on the gel (a thin
layer of
liquid is placed over the gel and, filtering through the gel into a
stack of
paper towels beneath, it transfers nucleic acids onto the filter under
the gel).
This "descending" transfer, as opposed to the widely used "ascending"
Southern
transfer, reduces the transfer time (to about 1 h) with equal or higher
quality
of the hybridization signal. The comparison of transfer kinetics by the
both
methods shows that (a) the Southern transfer of large size DNA fragments
proceeds quicker than it has been thought so far and is almost complete
within 4
h; (b) the descending transfer has an advantage over the ascending one
in the
rate of transfer (1-2 h) and its efficiency; and (c) the time of
transfer may
become a critical parameter upon using a filter with an apparently low
retention
capacity (Hybond N, Amersham) that is manifested by a decreased signal
at longer
than optimal transfer times.

PMID: 1706564, UI: 91174183



7: Biotechniques 1993 Jun;14(6):932-5

Effects of staining of RNA with ethidium bromide before electrophoresis
on
performance of northern blots.

Ogretmen B, Ratajczak H, Kats A, Stark BC, Gendel SM

Department of Biology, Illinois Institute of Technology, Chicago.

Staining of RNA with ethidium bromide (EtdBr) prior to running agarose
gels has
been reported to afford certain advantages over staining gels after
electrophoresis. We have examined prior staining of RNA with a wide
range of
EtdBr concentrations, particularly with respect to its effects on
Northern blot
hybridizations using antisense RNA probes. Prior staining with EtdBr at
concentrations of 100-1000 micrograms/ml caused significant alterations
in RNA
mobilities and significantly decreased hybridization with antisense RNA
probes
compared with unstained RNA. Prior staining with EtdBr at 10-50
micrograms/ml
resulted in the best combination of staining sensitivity, absence of
alterations
in RNA mobility and efficiency of hybridization. Conventional staining
of gels
after electrophoresis also resulted in decreased hybridization
efficiency with
RNA probes compared with unstained RNA.

Publication Types:
Technical report

PMID: 7687448, UI: 93326370



8: Anal Biochem 1994 Sep;221(2):303-5

One-hour downward capillary blotting of RNA at neutral pH.

Chomczynski P, Mackey K

Division of Endocrinology, College of Medicine, University of
Cincinnati, Ohio
45267-0547.

This report describes a setup for the downward capillary blotting of RNA
with
the use of 10 x SSC as a transfer solution. The setup is composed of a
stack of
blotting papers, hybridization membrane, and agarose gel. Two layers of
blotting
paper connect the stack with two reservoirs containing transfer
solution. Using
this setup, blotting of RNA fragments (< 7.5 kb) can be completed in 1
h. If
necessary, the blotting time can be expanded from 1 to 18 h without
decrease in
hybridization efficiency of RNA.

PMID: 7529006,

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