NorthernBlot--northern杂交

Preparation of Formaldehyde Agarose Gel

The gel conditions (1% agarose, 1X MOPS, 6.3% formaldehyde) are designed for ~4 hours of electrophoresis. If longer times are necessary, the formaldehyde concentration should be increased.

1.Heat agarose in water to get the agarose into solution.

2.Cool the agarose to approximately 55o C.

3.Add the appropriate volume of 10X MOPS and formaldehyde for final concentrations of 1X and 6.3%, respectively.

4.Pour the gel in the hood to avoid toxic formaldehyde fumes.

Preparation of RNA Samples

Generally, 10-30 μg of RNA is loaded per lane.

1.Add the appropriate volume of RNA to an eppendorf tube.

2.Dry down the samples in a Speed Vac.

3.Resuspend the samples in 25 μl of sample buffer and heat at 65oC for 10 minutes.

4.Add 2 μl of the dye solution and load gel.

Running Conditions

Run at 3.5 volts/cm for 3-4 hours, with recirculating buffer (1X MOPS). It is also acceptable to place the gel apparatus on two stirring blocks to recirculate the buffer. However, under these conditions it may be necessary to weigh down the gel with cheesecloth to prevent the gel from floating off the gel running platform.

Transfer of RNA to Membrane

1.After electrophoresis is complete, wash the gel twice in a large volume of deionized water to remove the formaldehyde.

2.Transfer the RNA by capillary blotting to nitrocellulose using a 20X SSC reservoir. Do not rinse the membrane after transfer.

3.Cross link RNA to nitrocellulose using the Stratalinker.

Hybridization Conditions

1.Use 100 μl of hybridization solution per square cm of membrane.

2.Prehybridize at 42℃for 1-2 hours in hybridization solution (see below) containing freshly boiled salmon sperm DNA at a final concentration of 100 ug/ml, and 1 ml of 50% dextran sulfate per every 5 ml of hybridization solution.

3.Add boiled probe to the prehybridization and continue 42℃incubation overnight.

Washing Conditions

Usually, two washes in 2X SSC, 0.1% SDS for 10 minutes each at room temperature is sufficient. However, if necessary, washes in 0.1 X SSC, 0.1% SDS 10 minutes at room temperature or 42℃may be done to reduce background hybridization.

Solutions

10X MOPS Buffer (store at 4oC in the dark)

0.2 M MOPS, pH 7

50 mM Sodium acetate

10 mM EDTA

Sample Buffer

50% formamide

2.2 M formaldehyde

1X MOPS

10X Running Dye Solution

50% glycerol

0.3% Bromphenol Blue

0.5 ug/ul Ethidium bromide

Salmon Sperm DNA

10mg/ml in water