EZQuant–ELN Knowledge Base

Northern Blotting	Table of Contents

Electrophoresis:

• Clean the gel box with NaOH and/or SDS, 2 hours to overnight, rinse with water.
• Prepare Agarose gel solution (1% Agarose, 1xMOPS, ddH₂O to 95% of end volume).
• Microwave until completely dissolved.
• Cool down to 50-60°C, add Formaldehyde (37%) to 0.6M end concentration and pour immediately. allow gel to harden at least 30 min.
• Prepare running buffer (0.2M Formaldehyde in MOPS).

Sample preparation:

• Use 5-10µg total RNA per lane (up to 30µg).
• Bring RNA samples to equal volumes (5-10µl) with DEPC-H₂O and add equal volume of loading buffer.
• Add 0.5µl ethidium bromide (0.5µg/µl).
• Heat for 5 min at 90°C, then cool on ice.

Running the gel:

• Run the gel (8x10cm) in fume hood at 70-100V (> 50-70mA)

Northern transfer of RNA:

• Soak the gel 3 x 5 min in ddH₂O (for removal of formaldehyde).
• Photograph the gel with a ruler beside it.
• Cut a membrane to the exact gel size.
• Soak the membrane in ddH₂O for a few seconds.
• Set up a capillary blot with 10x SSC transfer buffer: 2 wet Whatman papers - gel - membrane - 2 wet Whatman papers - 2 dry Whatman papers - paper towel - glass plate - weight.
• Transfer for 16-24 hr with changes of the paper towel.
• Mark lanes, remove the membrane and wash briefly in 2x SSC.
• Place the membrane on a wet Whatman paper and UV-crosslink damp.
• Bake the membrane at 80°C for 1-2 hr.

Hybridization:

• Pre-hybridize the membrane for 1-4 hr at 42°C with 5-10ml pre-hybridization buffer.
• Heat radioactive labeled probe for 3 min at 95°C, then cool on ice.
• Discard the pre-hybridization buffer, add hybridization buffer and probe and incubate overnight at 42°C.
• Wash the membrane for 15 min with 2x SSC at room temperature.
• Wash 2 x SSC+0.1% SDS at 65°C until background is low.
• Wash with 0.1x SSC+0.1% SDS at 65°C (optional).
• Expose wet membrane under saran wrap (-80°C).
  Important: never let the membrane dry (until the blot is stripped).