Northern Blot With Hybridization Bottles
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IMPORTANT: Once you have started this set of protocols, never let the membrane dry at any point (not even after it has been stripped). If the membrane is allowed to dry, it cannot be stripped. If handled properly, the membrane can be stripped and reprobed up to 14 times.
Hybridization Solutions:
Low Stringency Buffer
2X SSC
0.1% SDS
High Stringency Buffer
0.1X SSC
0.1% SDS
20x SSC (1L)
3M NaCl
0.3M Sodium Citrate
Adjust pH to 7.0.
Bring up to 1L.
Autoclave if needed. Store at room temp.
Detection Solutions:
Maleic Acid Buffer
0.1 M Maleic acid
0.15 M NaCl
pH to 7.5 with solid NaOH
Store at room temperature.
Blocking Solution
Dilute 10x Blocking Solution 1:10 with Maleic Acid Buffer.
Prepare fresh.
Washing Buffer
0.1 M Maleic acid
0.15 M NaCl
pH to 7.5
0.3% Tween 20
Store at room temperature.
Antibody Solution
Centrifuge Anti-Digoxygenin-AP for 5 min at 10krpm in its original vial prior to each use.
Pipet the necessary amount carefully from the top surface.
Dilute Anti-Digoxygenin-AP 1:10 000 (75mU/ml) in Blocking Solution.
Prepare fresh. (2 hrs, 40°C)
Detection Buffer
0.1 M Tris-HCl
0.1 M NaCl
pH to 9.5 (20°C)
Store at room temperature.
Ready-to-use CSPD (1ml)
20 µl CSPD Stock
980 µl Detection Buffer
Hybridization
Determine how much DIG Easy Hyb you will need for the procedures below. You will need enough buffer to completely cover the membrane during incubations. For 100cm² of membrane, you will need:
a. 20 ml for prehybridization
b. 5 ml for hybridization
I. Prehybridization
1. Prewarm buffer: Place the correct amount of DIG Easy Hyb in a sterile tube. Place the tube in a 50°C water bath.
2. Place the blot membrane into a prewarmed hybridization bottle.
3. Add the appropriate amount of prewarmed DIG Easy Hyb to the bottle.
4. Remove air bubbles from between the membrane and the bottle wall.
5. Incubate the blot for ½ to 3 hours at 50°C in a hybridization chamber. Rotate the bottle during the prehybridization. Be sure the bottle is balanced in the chamber with another bottle.
During the prehybridization (step 6), prepare the hybridization solution:
1. Determine how much DIG-labeled DNA probe you need (20-50ng probe/ml buffer).
2. Place the appropriate amount of probe in a microfuge tube with 50 µl of TE.
3. Place the tube in a boiling water bath for 5 minutes.
4. Immediately transfer the tube to an ice bath. Chill for 2 minutes.
5. Centrifuge the tube briefly.
6. Immediately add the denatured probe to a tube containing the appropriate amount of prewarmed DIG Easy Hyb (5 ml per 100cm²) and mix by inversion.
II. Hybridization
1. Discard (or save) the prehybridization buffer and add the hybridization solution to the bottle. Remove air bubbles from between the membrane and the bottle wall.
2. Incubate the blot for 6 to 16 hours at 50°C in a hybridization chamber rotating.
III. Stringent Wash
1. Discard (or save) the hybridization solution. Add 30mL of Low Stringency Buffer to the bottle.
2. Incubate the bottle at room temperature for 5 minutes with rotation.
3. Preheat 30 mL of High Stringency Buffer to 50°C.
4. Pour off the used buffer and immediately cover the membrane with fresh low Stringency Buffer.
5. Continue rotating at room temp for an additional 5 minutes.
6. Pour off the Low Stringency Buffer.
7. Immediately add the preheated High Stringency Buffer to the bottle. Incubate the blot twice (2x 15 min, rotating) in High Stringency Buffer at 500C. * If the probe is <80% homologous to the target, you need to perform these washes at a lower temp.
Detection*
1. Discard the High Stringency Buffer and add 50ml of Washing Buffer.
2. Incubate 2 min at room temp rotating.
3. Discard the Washing Buffer and add 30 ml of Blocking Solution.
4. Incubate membrane for 30 min (up to 3 hrs is ok) rotating.
5. Discard Blocking Solution and add 10ml of Antibody Solution.
6. Incubate membrane for 30 min rotating.
7. Discard the antibody solution.
8. Wash membrane twice (2 x 15 min) with 30ml portions of Washing Buffer.
9. Equilibrate membrane 3 min in 20ml Detection Buffer.
10. Place the membrane (RNA side up) inside a development folder (acetate sheets), hybridization bag, or other tightly sealable envelope-like container. (Do not use plastic wrap)
11. Wear gloves. For every 100cm² of membrane, apply 1ml (20-30 drops) of Ready-to-use CSPD (chemiluminescent substrate for alkaline phosphatase), dropwise, over the surface of the blot until the entire surface is evenly soaked. As you are applying the substrate, immediately cover the dampened part of the membrane with the second side of the container so the substrate is spread evenly over the membrane. Do not let air bubbles get trapped between the membrane and the upper surface of the container.
12. Incubate the membrane for 5 min at room temp.
13. Squeeze excess liquid out of the container and seal the sides of the container close to the membrane.
14. If using CSPD, incubate for 10 min at 37°C to enhance the luminescence reaction. If using CDP-Star, skip this step.
15. Expose the sealed envelope (containing membrane) to luminescence optimized X-ray film (15-25 min).
16. Based on the results in step 15, adjust the exposure time to get a darker or lighter band pattern. Repeat exposures can be made up to two days after adding the substrate.
Transparency Technique (alternative to steps 11-13):
1. Place the membrane on a sheet of transparency film.
2. Cover the membrane with dilute chemiluminescent substrate in Detection Buffer. For each 100cm² of membrane, use 500 µl of one of the following:
Ready-to-use CSPD
1:100 dilution of 25 mM stock CSPD
undiluted 0.25 CSPD ready-to use
1:200 - 1:500 dilution of 25 mM stock CDP-Star
1:2 - 1:5 dilution of 0.25 mM CDP-Star ready-to-use
3. Cover the damp membrane with a second sheet of transparency film.
4. Incubate 5 min.
5. Let excess liquid drip off membrane and heat seal the transparency sandwich around the damp membrane.
Stripping **
1. Rinse membrane thoroughly with double distilled water.
2. Incubate membrane twice (2 x 60 min) at 80°C in Stripping Buffer (50% deionized formamide, 5% SDS, 50mM Tris-HCl, pH 7.5) in a sealed bag.
3. Rinse membrane 5 minutes in 2x SSC at room temp.
4. Membrane can be reprobed immediately or stored wet in 2x SSC.
Notes
* Unless indicated otherwise, all of the incubations in this section are performed at room temp with shaking.
The volumes suggested are for a 100 cm² blot. Volumes can be adjusted according to the size of the blot and container. Be sure each solution completely covers the membrane and keeps it from sticking to the container as it shakes.
The transparency technique, which conserves chemiluminescent substrate, can be used as an alternative to steps 11 through 13. You can find the directions at the end of this section.
Always wear gloves when handling CSPD or CDP-Star.
** Always prepare Stripping Buffer just before use.
