Northern Blotting步骤和试剂配制(Breeden Lab)

Northern Blotting技术专题

I. Electrophoresis

clean gel box with NaOH and/or SDS, 2 hours to overnight, rinse with water

prepare Agarose gel solution [1 % Agarose, 1 x MOPS, H2O to 95 % of endvolume]

microwave until completely dissolved

cool down to 60-70 °C, add Formaldehyde (37 %) to 0.6 M endconcentration, pour immediately

allow gel to harden at least 30 min

prepare running buffer [1 x MOPS, 0.2 M Formaldehyde]

II. Sample preparation

use 5-10 µg total RNA per lane (up to 30 µg)

bring RNA with H2ODEPC to equal volume (5-10 µl), add same vol. loading buffer

add 0.5 µl EtBr (0.5 µg/µl)

heat for 5 min @ 90 °C, cool on ice

III. Gel run

run gel (8 x 10 cm) in fume hood with 70-100 V (-> 50-70 mA)

run until BPB is near the gel end (2.5-3.5 h)

IV. Northern transfer of RNA

soak gel 3 times 5 min in distilled water (to remove Formaldehyde)

photogragh gel with ruler beside it

cut GeneScreen membrane (Nylon, DuPont) to exact gel size

soak membrane in water for a few seconds

set up capillary blot with 10 x SSC transfer buffer:

2 wet Whatman - gel - membrane - 2 wet Whatman - 2 dry Whatman - papertowel - glasplate - weight

transfer 16-24 h with changes of the papertowel

mark lanes, remove membrane, wash briefly in 2 x SSC

place membrane on wet Whatman paper and UV-crosslink damp (auto crosslink setting, 254 nm, Stratagene, Stratalinker)

bake membrane @ 80 °C for 1-2 h

V. Hybridization

prehybridize membrane for 1-4 h @ 42 °C with 5-10 ml prehybridization buffer

heat radioactive labeled probe for 3 min @ 95 °C, cool on ice

discard prehybridization buffer, add hybridization buffer and probe, incubate ON @ 42 °C

wash membrane 1 x 15 min with 2 x SSC @ RT

wash with 2 x SSC, 0.1 % SDS @ 65 °C until background is low

wash with 0.1 x SSC, 0.1 x SDS @ 65 °C (optional)

expose wet membrane under saran wrap (-80 °C)

important: never let the membrane dry (until the blot is stripped)

VI. Stripping and re-hybridization

wash membrane for 30 min to 3 h in strip solution @ 75 - 85 °C until no radioactivity can be detected on the membrane

membrane can now be air dried and stored @ RT

for re-hybridization (up to 10 times) follow the hybridization protocol

Buffers:

10 x MOPS:

0.4 M Morpholinopropanesulfonic acid (free acid); 0.1 M Na-acetate-3 x H2O; 10 mM EDTA ; adjust to pH 7.2 with NaOH; store dark in fridge:

[500 ml: 41.9 g MOPS, 6.8 g NaAc, 10 ml 0.5 M EDTA]

Loading Buffer:

1 x MOPS; 18.5 % Formaldehyde; 50 % Formamide; 4 % Ficoll400; Bromophenolblue; store at -20 °C:

[1 ml: 100 µl 10 x MOPS, 500 µl Formamide, 185 µl Formaldehyde, 40 mg Ficoll400, Bromophenolblue, 215 µl H2O]

Prehybridization-buffer:

5 x SSC; 50 % Formamide; 5 x Denhardt"s-solution; 1 % SDS; 100 µg/ml heat-deNature d sheared non- homologous DNA (Salmon sperm DNA or yeast tRNA)

[100 ml: 25 ml 20 x SSC, 50 ml Formamide, 5 ml 100 x Denhardt"s, 1 g SDS, 1 ml 10 mg/ml DNA]

Hybridization-buffer:

Prehybridization buffer with 5 % Dextransulfate (Na-salt, MW 500,000, 50 % stock-solution) and without non-homologous DNA

100 x Denhardt"s solution:

[for 500 ml: 10 g Ficoll 400; 10 g polyvinylpyrrolidone MW 360000; 10 g BSA fraction V; H2O]

store at -20 °C.

20 x SSC:

3 M NaCl; 0.3 M Na-citrate

[1 l: 175.3 g NaCl, 88.2 g NaCitrate]

Strip-solution:

5 mM Tris pH 8; 0.2 mM EDTA; 0.05 % Na-pyrophosphate; 0.1 x Denhardt"s solution

[500 ml: 2.5 ml 1 M Tris, 200 µl 0.5 M EDTA, 5 ml 5 % NaPP, 1 ml 50 x Denhardt"s]