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Protocol 5.3: RNA Gel: A Denaturing Formaldehyde Gel

Protocol 5.3: RNA Gel: A Denaturing Formaldehyde Gel

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208



RNA PURIFICATION AND NORTHERN BLOT ANALYSIS



5. Add formaldehyde to give a final concentration of 6%. For this gel, add

29.2 ml of 36% formaldehyde (the concentration of the commercially

available stock is 36%). Mix thoroughly, but be careful to avoid creating

bubbles that are easily trapped in high-percentage gels.

6. Keep the agarose-formaldehyde gel at 65~ until the gel is poured. For

gel samples, load 3/~g total RNA per 1-cm-wide slot in the gel.

If necessary, dry down the RNA sample using a Speedovac and resuspend the sample in DEPCotreated H20. To prepare RNA samples for the

formaldehyde gel:

1. Mix

9 4.67 ~1 RNA sample

9 2/~1 10x Hepes buffer

2. Vortex.

3. Add 10/~1 deionized formamide and 3.3/~1 formaldehyde.

4. Mix well.

5. Incubate the RNA samples at 65~ for 10 min. This heat treatment will

disrupt the base pairing of any secondary structure of the RNAs.

6. Place the RNA samples on ice.

7. Add 4 ~1 RNA stop mix.

9 RNA stop mix or gel loading dye: 50% glycerol, 0.1% bromophenol

blue.

Gels typically are run at 100 V with a current of about 25 mA for

8-10 hr using Hepes buffer as the gel running buffer. After gel electrophoreo

sis, cut off any lanes to be stained from the gel and "blot" the rest of the

gel as a Northern blot.

To stain a formaldehyde gel, soak the gel in an ethidium bromide

solution (about 0.5 /~g/ml) for about 20 min. Destain the gel for about

2 hr. Monitor the progress of the destaining. A gel may need to restained

or destained longer.

To deionize formamide, add approximately I g of Dowex MR-3 mixed

resin beads to about 100 ml of formamide. Mix well and let the sample

incubate at room temperature for at least 30 min before the formamide is

used. Store the deionized formamide tightly covered, in the dark, for up

to 1 week. Alternatively, purchase molecular biology grade formamide

which can be used as is. Store aliquots of high-quality formamide at - 20~



PROTOCOL 5.4:

A Northern Blot

For a Northern (reverse Southern) blot, RNAs are subjected to electrophoresis in a denaturing agarose gel, such as a formaldehyde gel, a glyoxal



PROTOCOL 5.4: A NORTHERN BLOT



209



gel, or a methyl mercury gel. When the RNAs have migrated far enough,

the gel is ready to be blotted.

Procedure for Blotting a Gel



1. Obtain a glass baking dish, a glass plate large enough to hold the

gel, and 4 bottle caps (supports for glass plates). Wear gloves when

handling these. Wrap glass plate with a piece of Whatman (No. 1)

chromatography paper cut to surround the glass plate completely. Put

the glass plate on supports in the baking dish.

2. Pour 20• SSC (3 M NaC1, 0.3 M Na3 citrate, pH 7) into the baking

dish, wetting the filter paper. The level of 20• SSC should not quite

reach the glass plate. The filter paper will act as a wick.

3. Place the gel on the wetted filter paper on glass plate. Mark a gel

corner with a small notch to indicate the orientation of the gel. (A

horizontal gel should be placed on the Whatman filter paper upside down so the nitrocellulose paper will contact the bottom of the

gel.)

4. Cut a piece of nitrocellulose paper to fit the gel exactly. Gloves should

be worn when handling the nitrocellulose paper. Cut the nitrocellulose paper with a very sharp razor blade; a dull blade will tear the

nitrocellulose paper.

5. Wet the nitrocellulose paper with distilled H20: lay paper on top of

a tray of H20 and allow it to soak; do not submerge the paper. When

one side is wetted, flip the paper over to wet the other side.

6. Once the nitrocellulose is totally wetted, soak paper in 20• SSC.

7. Place the nitrocellulose paper on top of the gel, taking care to position

the paper precisely. Be especially careful to line up the top of the

paper with the top of the gel (wells). Also take great care to avoid any

air bubbles between the gel and the paper.

8. Cut 4 to 5 pieces of Whatman filter paper exactly to the size (or just

slightly smaller) of the nitrocellulose paper. Wet 2 pieces of filter

paper in 20 • SSC and place on top of the nitrocellulose paper. Avoid

air bubbles. Then place 2 to 3 pieces of dry filter paper on top. DO

NOT allow paper to hang over edge so that it might act as a wick.

9. Cut a stack of paper towels _~ 2 in. high just to fit or be slightly smaller

than the nitrocellulose paper. Place a weight, such as another glass

plate, on top of the paper towel stack.

10. Allow gel to set overnight, adding more 20x SSC as needed to keep

the SSC level just at the bottom of the glass plate. Change paper towels

as needed.

11. Blot the gel overnight.

12. Mark the top edge of the nitrocellulose paper with indelible ink.



210



RNA PURIFICATION AND NORTHERN BLOT ANALYSIS



Procedure for a Northern Blot

DO NOT WASH THE BLOT. If the blot is to be cut into strips, this

should be done with a sharp razor while the nitrocellulose paper is

still wet.

13. Pat the blot dry on paper towels.

14. Bake the blot in a vacuum oven at 80~ 2 - 4 hr. This fixes the nucleic

acid to the nitrocellulose paper.



PROTOCOL 5.5:

Standard Northern Blot Hybridization Conditions

for 32P-Labeled Probe

Materials

Northern Prehybridization Solution

Final concentration

of component



Concentration of

stock component



50% formamide

5x SSC

50 mM PB

5x PM

100/~.g/ml sheared, denatured

calf thymus DNA

0.1% SDS



Formamide

20x SSC

1M PB

lOOx PM

2 mg/ml sheared, denatured

calf thymus DNA

20% SDS



H20



Amount of stock

component to add

5 ml

2.5 ml

0.5 ml

0.5 ml

0.5 ml

50/zl

0.95 mlmto a total volume of

10 ml



Northern Hybridization Solution

Final concentration

of component

50% formamide

5x SSC

20 mM PB

lX PM

100/zg/ml sheared,

denatured calf thymus

DNA

5% dextran sulfate



Concentration of

stock component

Formamide

20x SSC

1M PB

100 x PM

2 mg/ml sheared,

denatured calf thymus

DNA

50% dextran sulfate

H20



Amount of stock

component to add

5 ml

2.5 ml

0.2 ml

0.1 ml

0.5 ml



1.0 ml

1.1 ml--to a total volume

of 10 ml



Washes for Northern blot: l x SSC, 0.1% SDS, 5 mM EDTA.



PROTOCOL 5.5: STANDARD NORTHERN BLOT HYBRIDIZATION CONDITIONS



211



NOTES



1. Use molecular biology grade formamide or deionize the formamide

before using. To deionize formamide, add approximately I g of Dowex

mixed resin beads to approximately 100 ml of formamide. Mix thoroughly. Store in the dark or wrap the bottle with aluminum foil. Let

the formamide and beads stand for several hours before using.

2. SSC is standard sodium citrate, lX SSC is 8.8 g NaC1, 4.4 g Na citrate/

liter, pH 7.0.

3. PB is phosphate buffer. 1 M PB is pH 6.8. Make 1 M PB by mixing

equal volumes of I M monobasic sodium phosphate and dibasic sodium

phosphate.



100 x Denhardt's solution:

2% (w/v) BSA (bovine serum albumin)

2% (w/v) PVP (polyvinylpyrrolidone, molecular weight (MW)

4 x 10 4)

2% (w/v) Ficoll (MW 4 x 105, a nonionic synthetic polymer of

sucrose)

These three components are "nonspecific blockers." They help

decrease "background" of nonspecific binding of probe to nitrocellulose membrane.

4. PM is prehybridization mix or Denhardt's solution.

5. SDS is sodium dodecyl sulfate.

6. Dextran sulfate is a high-molecular-weight (MW 500,000) synthetic

polyanion. It is added to increase the rate of hybridization of the probe

via an excluded volume effect, effectively increasing the concentration

of the probe in solution.

CAUTION: Before working with 32p, review the radiation safety rules

and disposal protocols for your school.



Procedure

1. Seal the baked nitrocellulose blot in a Seal-a-Meal plastic bag containing 10 ml of Northern prehybridization solution.

2. Incubate the blot in prehybridization solution for 10 to 15 hr or overnight at 42~

3. Open the plastic bag by cutting open a corner. Pour out the prehybrido

ization solution. Add 10 ml Northern blot hybridization solution.

Reseal the plastic bag.

4. Incubate the blot in hybridization solution for 4 to 6 hr.

CAUTION: When working with radioactivity, wear double layers of

gloves, a laboratory coat, and goggles. Work in an area designated for



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