生物行移动版

Tuesday, September 30, 2003

Description

Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF
"Horowitz, R. and Frank, M. B. Northern Blots. In: Frank, M. B. ed. Molecular Biology Protocols . (http://omrf.ouhsc.edu/~frank/nblots.html). 1997. Oklahoma City. Revision Date: October 2, 1997."

Procedure

Sample Preparation, Electrophoresis, and Transfer

Prepare a 1% agarose gel in DEPC treated water. Cool for about 10 minutes and add MOPS diluted from a 20X MOPS buffer stock to 1x. Make the final solution 0.6% formaldehyde in a fume hood. Transfer solution to an RNA electrophoresis tank and allow the gel to solidify. (For example, for the Hoeffer HE33 blue gel apparatus kept at -20C, prepare 50 ml agarose by combining 0.5 g agarose, 46.8 ml DEPC treated H 2O, 2.5 ml DEPC 20x MOPS, 0.8 ml 37% formaldehyde.

Prepare RNA: For total RNA, use at least 10 g of RNA per lane. For poly-A+ RNA, use at least 1-2 g of RNA per lane. Keep the volume of RNA below 20 l. QS to 20-30 l with sample buffer. Incubate at 65C for 5 minutes. Add 1/20 volume RNA stop dye and 1 l of 1 mg/ml ethidium bromide. Load on gel.

Electrophorese in 1x MOPS buffer with a circulating pump at 40-60 constant volts for about 2-3 hours. Stop the electrophoresis when the stop dye has migrated about 2/3 - 3/4 of the way through the gel. Soak the gel in 200 ml RNase-free water for 10-15 min and then for 15 min in 50 mM NaOH. Neutralize the gel by soaking in 10x SSPE for 30 min.

Transfer RNA overnight to a nylon membrane (pre-wet in 1x MOPS buffer) with a 10x SSPE stock solution (see Southern blot protocol). Mark the position of the wells on the blot and label it (pencil is fine). Rinse in 4x DEPC treated SSPE and UV treat for 5 minutes. If you are studying whole cell RNA, the position of the rRNA bands may be marked in pencil on the UV light box. 28s rRNA is about 4800 bases and 18s rRNA is about 1900 bases.

(optional): Prehybridize the blot in 1x SSPE, 0.1% SDS at 65C for 1 hour. Store in Saran wrap at 4C for future use.

Hybridization and Washing:
Conditions here are typically empirically determined depending on the background and levels of particular transcripts in the cells being studied.

Prehybridize the membrane at 42C in 50% deionized formamide, 5x SSPE, 50 mM sodium phosphate buffer, pH 6.8, 1x Denhardt's solution, heat denatured salmon sperm DNA to 100-200 g/ml, and 0.5% SDS for 3-4 hours.

Hybridize with radiolabelled probe overnight at 42C in fresh prehybridization buffer with 7.5% dextran sulfate. We label 25 ng of a DNA probe with random primers and use it all. In a rotating cylinder air incubator, about 5 ml of hybridization solution is sufficient.

The next day, wash the membranes twice in 2x SSPE, 0.1% SDS at room temperature, 10 minutes per wash, then for 30 minutes in 0.5x or 0.2x SSPE, 0.1% SDS at room temperature. Check the membrane for radioactivity with a hand-held monitor and/or autoradiography before increasing the stringency. Try a test exposure for about 48 hours.

Recipes

20x MOPS (500 ml)
41.9 g MOPS
6.8 g sodium acetate (mw 136.08)
2.6 g EDTA (mw 372.24)
400 ml DEPC H 2O
Adjust pH to 7.0 with NaOH and QS to 500 ml with DEPC-treated
H 2O.

RNA Sample Buffer
50 l deionized formamide
15 l formaldehyde
5 l 20x MOPS

20x RNA Stop Dye
1x MOPS
50% glycerol
0.1% bromophenol blue

Supplies

Tips

NOTE:Treat all concentrated buffer stocks with 0.1% DEPC overnight in the hood before autoclaving. Autoclaving will turn the MOPS buffer yellow.