Northern Blot

From ChoanoWiki

DAY 1

Decontaminate the gel box:

• Soak gel box, casting trays, combs, Erlenmeyer flask and 100mL graduated cylinder in 0.1N NaOH, 1mM EDTA for at least 20 minutes and rinse with MQ water

Make/pour the gel:

100mL gel (1.2%)

• 1.2g agarose
• 73mL DEPC H20
• 10mL 10x MOPS
• 17mL 37% formaldehyde

125mL gel (1.2%)

• 1.5g agarose
• 91.25 mL DEPC H20
• 12.5mL 10x MOPS
• 21.25mL 37% formaldehyde

• Melt the agarose in the water and allow to cool to 60-65 C (can be done in water bath)
• Mix the MOPS and formaldehyde in the graduated cylinder
• Gradually add the MOPS/formaldehyde mix to the melted agarose. Swirl and mix well.
• Pour the gel in the hood. Remember, the thinner the gel, the more efficient the transfer will be.

Prepare the RNA samples:

• The RNA should be resuspended in RNase free water
• Use 10-20 μg of total RNA per lane depending on the expression level of the transcript you are probing for
• Prepare your RNA ladder in the same way as your RNA samples but do not heat the ladder

To each sample add:

• 5μl formamide
• 1x MOPS
• 4% formaldehyde

• This can be made as a master mix and then 8.5μl added to each sample
• Heat the RNAs to 65 C for 5 minutes. Move to ice.
• Make a dye solution containing 4 parts bromophenol blue/xylene cyanole dyes:1 part stock ethidium bromide (1mg/mL)
• Add 2.5μl of the dye mix to each sample
• Load the entire sample into your gel
• NOTE: Both the master mix and the dye solution should be added to the RNA ladder but it should not be heated

Run the gel:

• Run the gel at 70 Volts (5 V/cm or less) until the dye front is close to the bottom of the gel
• The running buffer is 1x MOPS

The transfer:

• Cut 5 sheets of Whatman paper, one sheet of Zeta-Probe membrane and a 15cm stack of paper towels to the exact size of the gel.
• Soak the membrane in MQ water for five minutes while setting up for the transfer
• Place sponge(s) in the bottom of a glass dish. The sponge(s) must be larger than the gel
• Place enough 10x SSC buffer in the transfer dish to immerse the sponge about half way up its side
• Place 3 sheets of Whatman paper on the sponge and flood the paper surface with buffer. Roll a pipette over the paper to remove bubbles
• Place the gel on top of the paper and remove any bubbles by pressing them away.
• Spread plastic wrap over the gel and cut out a window with a new razor blade allowing only he gel to emerge through the window.
• Flood the surface of the gel with buffer and place the pre-wetted membrane on top of the gel, being sure to line up the top of the gel with them membrane. Remove bubbles by rolling a pipette over the membrane.
• Flood the membrane with buffer. Wet 2 sheets of Whatman paper in 10x SSC and then place them over the membrane. Be they are aligned with the membrane and completely cover it without touching the gel.
• Stack the paper towels on the Whatman paper
• Do not put excessive weight on top of the gel
• Keep an excess of buffer in the dish but so not cover the top of the sponge
• Allow the transfer to continue overnight

DAY 2

• Separate the membrane from the gel, rinse briefly in 2x SSC and blot with filter paper
• UV crosslink the RNA to the membrane and bake in the oven at 80 C for 2 hrs

pre-hybridization and hybridization:

• Be sure that the hybe tubes have been decontaminated with NaOH, EDTA solution
• Wet membrane in 6x SSC
• Place membrane RNA-side-up in hyb tube and add 10ml of pre-hyb solution
• Incubate in hyb oven for 2 hours at 65 C

During the blocking reaction label the probe:

Combine:

• 100ng DNA template
• 1ul random primers (125ng/ul)
• DEPC H20 to 30ul

• Boil at 100 C for 2 minutes
• Incubate on ice for 2 minutes
• Spin quickly
• Return to ice

Add to the mix:

• 1ul 5mM dNTP solution
• 10ul 5x random priming solution
• 1ul 10mM DTT
• 5ul 10mCi/mL [α-32P] dCTP
• 2ul DEPC H20
• 1ul (5U) Klenow fragment

• Mix and spin
• ncubate at room temp for 1-2 hours

When labeling reaction is done:

(about 45 minutes before the blocking is finished)

• Clean labeling reaction with Illustra ProbeQuant G-50 Micro Column ***be sure to dispose of radioactive waste properly!!!!
• Boil probe for 10 min
• Transfer to ice and spin down
• Add probe to pre-hyb
• Incubate overnight at 65 C

DAY 3

Wash membrane: remember to discard all radioactive solutions in waste!!!! all washes done in hyb oven with rotation

• Prewarm 0.2x SSC, 0.1% SDS at 42 C and 0.1x SSC, 0.1% SDS at 68 C
• Pour off hyb solution
• Add 10mL 2x SSC, 0.1% SDS and incubate for 5 min at room temp
• Change wash solution and repeat
• Replace wash with 10mL 0.2x SSC, 0.1% SDS and incubate for 5 min
• Change wash solution and repeat
• Survey membrane with the geiger counter for background
• If needed, add 10mL 0.2x SSC, 0.1% SDS prewarmed to 42 C and incubate for 15 min at 42 C
• Change wash solution and repeat
• Survey membrane with the geiger counter for background
• If needed, add 10mL 0.1x SSC, 0.1% SDS prewarmed to 68 C and incubate for 15 min at 68 C
• Change wash solution and repeat
• Remove the final wash and rinse membrane in 2x SSC at room temp
• Blot excess liquid and cover with plastic wrap
• Perform autoradiography

Solutions

Pre-hybridization buffer

• 0.5 M Na2HPO4, pH 7.2
• 7% (w/v) SDS

20X SSC, pH7.0

• 175.3g NaCl
• 88.2g Sodium Citrate
• 800mL dH20

• Adjust pH to 7.0 with HCl
• Adjust volume to 1L