DIG Northern protocol (as requested by many)

Arnoud van Vliet avvliet at knoware.nl
Mon Oct 2 14:54:02 EST 2000


NORTHERN BLOT WITH DIG-LABELLED RNA/DNA PROBES

(Arnoud van Vliet (avvliet at knoware.nl), October 2000; satisfaction not
guaranteed)

Important things:
1. In my hands, only RNA probes work properly. Random primed DNA probes
might work with very strongly expressed messengers. PCR probes jus don't
work
2. Buy deionized formamide in small volumes (100 ml), and only use them for
Northerns. Same goes for formaldehyde. I personall use Sigma's stuff (no
affiliations).
3. We routinely clone the PCR fragment with the T7 promoter, and use the
clone for new amplifications. However, one probe preparation should suffice
for at least 10-20 probes, and each probe can be reused a few times by
heating to 70 degrees C.

PREPARING THE PROBE

Design primers for PCR amplification of desired fragment. To create an
antisense RNA probe, add to the reverse primer a 5' sequence containing the
T7 promoter:
5'-CTA-ATA-CGA-CTC-ACT-ATA-GGG-AGA

Example: Helicobacter pylori ureB primers:
Forward primer: 5'-ATGAAACTCACCCCAAAAGA
Reverse-T7 primer: 5'-CTAATACGACTCACTATAGGGAGA-GGAAGTGTGAGCCGATTTGA

PCR amplify in 50-100 ul volume, clean up with QiaQuick column.

Preparing an RNA probe
Use Boehringer's T7/SP6 in vitro transcription kit, mix:

1. Mix:
PCR product (0.5-1 ug, usually about 15-20 ul)
3 ul NTP/DIG-UTP solution
3 ul 10 x Dig reaction buffer
1 ul RNAse inhibitor (optional)
2 ul T7 RNA Polymerase
RNAse-free water to 30 ul

2. incubate for 2-3 hours at 37°C
3. add DNAse, RNAse free (2 ul DNAse I solution), incubate 15 min at 37 °C
4. stop reaction by addition of 2 µl EDTA (0.5 M, pH 8.0)
5. purify labeled RNA by precipitation with 4 M LiCl (3.2 ul) and 80 ul EtOH
6. incubate 15-30 min at -80°C
7. centrifuge 15 Min at 14,000 rpm
8. wash once with 70% EtOH
9. dry pellet briefly, resuspend in 100 ul RNAse-free water
10. Quantitate labelling as in DIG-manual, chapter 5.

1. PREPARATION AND RUNNING OF FORMALDEHYDE-AGAROSE GELS (1.5%)

1. Make tray, comb and tank RNAse-free by cleaning with soap, washing with
demi-water, and incubating for >30 min in 0.1-0.2 N NaOH, followed by
washing with RNAse-free water. Dry tray and comb, when necessary briefly
clean with 70% EtOH
2. Pour gel:

for: 100 ml (midi gel, max 20 lanes with 2 combs)
75 ml 2% agarose (cooled to 60-65 degrees C)
2 ml 50x Running buffer
5.3 ml formaldehyde 37%
17.7 ml aqua dest, RNAse-free
Pour directly after mixing, in fumehood

for: 125 ml (midigel, max 24 lanes with 2 combs)
93.25 ml 2% agarose (cooled to 60-65 degrees C)
2.5 ml 50x Running buffer
6.7 ml formaldehyde 37%
22.05 ml aqua dest, RNAse-free
Pour directly after mixing, in fumehood

Let set for minimal 30 min, put wrap over gel. Formaldehyde gels are not as
strong as corresponding normal agarose gels!

3. Make Running Buffer: dilute 50´ Running buffer in RNAse-free water.
Volume needed:
small midigel: 600 ml (12 ml 50´ Running buffer)
large midigel: 800 ml (16 ml 50´ Running buffer)

4. Prepare RNA samples:
Mix 2 ul RNA sample with 8 ul RNA denaturation mix
Incubate at 65°C for 15 min
Add 1 ul loading RNA loading buffer

5. Put gel in tank, submerge in buffer. Careful with slots, as gel is weaker
than normal agarose gel. Submerge in as little buffer as possible!

6. Load samples, and run gel at 50-80V until bromophenol blue band reaches
has run 3/4 of the gel.

NOTE: the sodium phosphate buffer used here can be treated with DEPC, but
its buffering capacity is much lower than that of MOPS. It is therefore
absolutely necessary to stop the gel every 30 min, reverse the gel and the
electrodes, otherwise the RNA close to the cathode is damaged and does not
give sharp bands anymore. Alternatively, circulate the buffer.


Materials

50 X Running buffer
0.9 M Na2HPO4
0.1 M NaH2PO4
(Treat with DEPC)
Autoclave

2% Agarose
2 g agarose in RNAse-free aqua dest
Autoclave

10x RNA loading buffer
50% Glycerol
1 mM EDTA
0.25% Bromophenol blue (Sigma)
0.25% Xylene cyanol (Sigma)
Treat with DEPC, autoclave

RNA denaturation mix (per 2 ml RNA sample)
0.16 ul 50´ Running buffer
1.75 ul formaldehyde 37%
5 ul formamide, deionized
1.09 ul RNAse free water

For 20 samples of 2 ml:
3.2 ul 50x Running buffer
35 ul formaldehyde 37%
100 ul formamide, deionized
21.8 ul RNAse free water


RNA BLOTTING

1. After running the formaldehyde gel, submerge gel in 10x SSC and put on
shaking platform for 2x 20 min at room temperature
2. Make standard blotting setup like standard Southern blot.
3. Blot overnight at room temperature
4. Wash blot for 1-2 min in 10x SSC
5. Dry briefly on 3 MM paper
6. Crosslink membrane at 120 J/cm2
7. Stain for 1 min with methylene blue
8. Destain by rinsing with RNAse free water
9. Make photograph/scan of blot
10. Store at -80°C or use directly in hybridization

Materials

10x SSC
1.5 M NaCl
0.15 M Sodium citrate
pH 7.0 with NaOH (when necessary)
Treat with DEPC, autoclave

Methylene blue staining solution (reusable)
0.3 % methylene blue in 0.3 M sodium acetate pH 5.2
Autoclave

NORTHERN HYBRIDIZATION

1. Prehybridize membrane at 65°C overnight. This might be essential!
2. Denature probe: 5 min 100°C in heating block. Final concentration in
hybridization: 10 ng/ml
3. Take aliquot of prehyb mix, and add denatured probe to aliquot. Then add
aliquot to rest of prehybmix with membrane. Don't add probe directly to
membrane!
4. Hybridize O/N at 65°C
5. Wash blot 2x 5 min at 65°C with low stringency wash buffer, and 2x 30 min
at 65°C with high stringency wash buffer
6. Wash blot 2x 5 min at RT with DIG-buffer. It is essential to wash away
all salts from hybridization
7. Block membrane in Blocking buffer for 30-60 min at RT
8. Incubate 30-60 min with anti-DIG-AP (1:10,000 diluted in Blocking buffer)
9. Wash 2x 15 min at RT with DIG-buffer
10. Equilibrate in AP-buffer
11. Do chemiluminescent detection with CDP-Star

Materials

(Pre)hybridization mixture
50% formamide, deionized
5x SSC
2 % blocking reagent
0.1 % sarkosyl
0.5 % SDS
200 ug/ml denatured herring sperm DNA

Wash buffers (prewarmed at 65°C)
(Low stringency):
1x SSC
0.5 % SDS
0.1 % sarkosyl
(high stringency):
0.1x SSC
0.5 % SDS
0.1 % sarkosyl

DIG-buffer
0.1 M Maleic acid pH 7.5
0.15 M NaCl
Treat with DEPC, autoclave
Add Tween-20 to 0.1%

Blocking buffer
DIG-buffer with 1% blocking solution

AP-buffer
100 mM Tris pH 9.5 (pH 9 is also OK)
100 mM NaCl
Autoclave








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