Northern Blotting

DNA Transfer

  1. Pour gel in hood.

  2. Mix RNA (10 ug) with 1.5X loading buffer.

  3. Incubate 5 minutes at 65oC. Cool on ice.

  4. Add 2 ul loading dye.

  5. Pre-run gel at 5 V/cm for 5 minutes. Load samples and run. Mix buffers after 3 hours.

  6. Wash gel 2X in water for 15 minutes, then 1X in 10X SSC for 15 minutes. Photograph gel under UV.

  7. Cut nylon membrane (MSI 0.45 micron #N04HY00010) and several pieces of blotting (e.g. Schleicher and Schuell GB002) paper to the same size as the gel. Wet the nylon with distilled H20, then soak in 5X SSC buffer.

  8. Assemble sandwich:

    1. Place down a large sheet of plastic wrap.

    2. On top of the plastic wrap, place two pieces of blot paper (precut to same size as gel) soaked in 20X SSC.

    3. On top of blot paper, place gel with wells-side down.

    4. On top of gel, place presoaked nylon.

    5. On top of nylon, place one piece of blot paper soaked in 5X SSC.

    6. On top of blot paper, place 10-15 more pieces of dry blot paper.

    7. Wrap whole sandwich in the plastic wrap.

    8. Place a glass plate and weight on top and let transfer for 3-18 hours.

  1. Crosslink afler blotting.

Hybridization and Detection

  1. Denature probe (dig-dUTP labeled) for 5 minutes at 90-100oC in boiling water bath. [Perform this step for newly labelled probes (in eppendorf tubes) or "used" probes already in hybridization solution (stored at -20oC in 50 ml conical tubes)].

  2. At the same time, prehybridize blot with at least 15 ml hybridization solution in a hybridization tube for at least 5 minutes. Any prehybridization time of 5 minutes or longer is adequate.

  3. If using a newly labelled probe, pipet probe into hybridization solution (not onto the blot). If reusing a probe, pour off the (pre)hybridization solution, save at 4oC for reuse; then dump the used probe/hybridization solution onto the blot. For newly labelled probes, try 10-15 ul of labeled DNA to 15 ml hybridization solution for hybridization.

  4. Incubate blot for at least 5 hours to overnight at 42oC.

  5. Pour off probe and save at -20oC in 50 ml conical tube.

  6. Wash blot with Blot Wash #1 twice at 5 minutes per wash at room temperature.

  7. Wash with Blot Wash #2 twice at 15 minutes per wash at 55oC.

  8. Wash with Buffer #1 for 1 minute at room temperature.

  9. Block with Buffer #2 for 30 minutes to overnight. After use, save and store Buffer #2 at 4oC.

  10. Add 10 ul antibody to 40 ml Buffer #1 and 10 ml Buffer #2. Incubate blot with antibody solution for 30 minutes. After use, save and store antibody solution at 4oC.

  11. To remove unbound antibody-conjugate, wash with Buffer #1 twice at 15 minutes per wash.

  12. Equilibrate blot with Buffer #3.

  13. Prewarm CSPD Ready-to-Use to room temperature

  14. Transfer blot carefully from Buffer #3 (let drip ~5 seconds) to a plastic tray.

  15. In the darkroom, incubate the blot with ~25 ml CSPD Ready-to-Use for ~1 minute. Dump solution back into a dark bottle.

  16. Drag blot along the edge of the tray to remove excess liquid, and place blot onto a plastic report cover.

  17. Blot around edges with a tissue and then "close" the plastic cover. Rub gently to remove bubbles.

  18. Expose to film. Incubate cassette at 37oC for 10 minutes, then return to room temperature. Exposures vary from blot to blot (1-2 hours).

    Note: CDP Star is another detection reagent from Boehringer-Mannheim (that may be used instead of CSPD Ready-to-Use). It is supposed to be ~10X more sensitive.

Stripping

  1. Wash blot in water for 2-5 minutes.

  2. Strip with 500 ml stripping solution (0.2N NaOH 0.1% SDS - made fresh) at 37oC for 30 minutes.

  3. Wash with 2X SSC at room temperature for 1 minute. Keep in 2X SSC until ready to use.

  4. Prehybridize for 5 minutes and hybridize to neXt probe as shown on Southern protocol.

Note: This solution should not be used with Northerns because of the NaOH.



Formaldehyde gel

For 200 ml gel: 

  • 1.8g agarose
  • 200 ml distilled H2O
  • 10 ml 37% formaldehyde stock solution
  • 20 ml 10X MOPS running buffer
After dissolving agarose in distilled H2O, let cool to 65oC before adding final reagents.



1.5X Loading buffer

  • 75 ul formamide
  • 750 ul 37% formaldehyde from stock solution
  • 150 ul 10X MOPS running buffer
  • 15 ul 5 mg/ml EtBr
  • 10 ul distilled H2O
Best results if made fresh each time.



Loading Dye

Bromophenol blue in 35% glycerol and 1X running buffer



Denaturation Buffer
(0.5 M NaOH, 1.5 M NaCl)

TO MAKE 20 L: 

  • 400 g NaOH
  • 1752 g NaCl
Fill to 20 L with distilled H2O.

Neutralization Buffer
(0.5 M Tris, pH 7.0, 3.0 M NaCl)

TO MAKE 20 L: 

  • 1211 g Tris
  • 3506 g NaCl
Adjust pH to 7.0 using concentrated HCl. Fill to 20 L with distilled H2O.

20X SSC
(3 M NaCl, 0.3 M sodium citrate, pH 7.0)

TO MAKE 5 L: 

  • 876.6 g NaCl
  • 441.15 g sodium citrate
Bring to pH 7.0 with concentrated HCl. Dilute as necessary to give 5X SSC and 2X SSC.

Hybridization Solution
(5X SSC, 0.5% (w/v) Blocking Reagent, 0.1% (w/v) N-lauroylsarcosine, Na-salt, 0.02% (w/v) SDS, 50% (w/v) formamide)

TO MAKE 40 ml: 

  • 10 ml 20X SSC
  • 0.2 g blocking reagent (supplied with kit)
  • 40 mg N-lauroylsarcosine, Na-salt
  • 40 ul 20% SDS stock solution
  • 20 g formamide
Blocking reagent does not dissolve rapidly. Heat to ~50-70oC; the solution remains turbid. Make ~1 hour in advance. Store hybridization solution at 4oC in a bottle or at -20o
Note: Prehybridization solution is the hybridization solution without the labelled probe.

Blot Wash #1 (2XSSC, 0.1%(w/v) SDS)

TO MAKE 500 ml: 

  • 50 ml 20X SSC
  • 2.5 ml 20% SDS stock solution


Blot Wash #2 (0.1X SSC, 0.1%(w/v) SDS)

TO MAKE 500 ml: 

  • 10 ml 5X SSC
  • 2.5 ml 20% SDS stock solution


Buffer #1 (100 mM maleic acid, 150 mM NaCl)

TO MAKE 20 L: 

  • 232.2 g maleic acid
  • 175.5 g NaCl
Adjust pH to 7.5 with 156 g NaOH.

Buffer #2 (2% blocking reagent, 100mM maleic acid, 150mM NaCl)

TO MAKE 500 ml: 

  • 10 g blocking reagent
  • 498 ml Buffer #1
Heat to 50-70oC; prepare ~60 minutes in advance; solution remains turbid
Store at 4oC. Add NaN3 for long term storage.

Buffer #3 (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2)

TO MAKE 500 ml: 

  • 6 g Tris
  • 2.9 g NaCl
  • 25 ml 1 M MgCl2 stock solution

NOTES:
All solutions should be at room temp when used on blots.

We reuse prehybridization solution, probes, Buffer 2, antibody conjugate, and CSPD Ready-to-Use. As needed, we replace the prehybridization solution, Buffer 2 and CSPD with fresh solutions. The probes and antibody solutions are used for several months and spiked or made fresh as needed. For most probes, only half of the labelling mixture is needed for sufficient signal. Recipes are from the Boehringer Mannheim Nonradioactive DNA Labeling and Detection Kit and protocols.

Miscellaneous Gottschling Lab Lore:
Fragments with incorporated alkali-labile dig nucleotides may be stripped from blots; the alkali-stable versions do not strip off well.


The blocking reagent (Buffer 2) seems to work better after a couple of uses. In the first couple of uses, background spots may be abundant.