Northern blot to quantitate 16S vs 17S
It is useful to be able to quantitate the ratio of 16S vs 17S rRNA within the cell, as accumulation of 17S rRNA can be indicative of ribosome assembly defects. It can be difficult to get good separation of the two species, however. This protocol produces reliable results.
You will need a fairly large-format agarose gel. We use a tray about 20 cm long. A mini-gel will not work.
For best results you will want to run the gel slowly overnight. If time is constrained you may run the gel at a higher voltage for shorter time, but the results will not be as pretty.
Make 1.5 L of 10 mM sodium phosphate buffer, pH 7.0
Pour a 1.4% 1:1 SeaKem to NuSieve agarose gel in 10 mM sodium phosphate buffer. Use a large gel tray. The one we use takes about 200 mL of gel. Use a wide-toothed comb.
Make up your samples. For total bacterial RNA, prepare ~2 ?g samples. You can use much less RNA for pure 16S or 17S control lanes.
To make up samples
5.4 µL RNA
5.4 µL glyoxal
16 µL DMSO
3 µL 100 mM sodium phosphate buffer
Incubate at 50°C for 1h
During the incubation it is wise to set up the gel box. This running buffer is very low in ionic strength so must be recirculated in some way. Obviously a recirculating rig can be used, but it is not required. Another simple method is to set the gel box on two stir plates, one under each end. Put a small stir bar into each buffer well and set them stirring. This is convenient for overnight runs. Alternatively the tray can be rotated 180° in the box every 30 min to prevent build-up of an ion gradient. Be sure to rotate the lid, too, so the positive electrode is always at the bottom of the gel! This method is more practical when doing a short run at high voltage.
At the end of the 1 hour incubation chill samples on ice
Add 4 µL glyoxal loading buffer. Vortex and spin down
Load onto gel.
Run at 130 V until the samples are into the gel–15 min or so. Turn the voltage down to 35-40 V and run ~16h. If you run the bromphenol blue to the bottom of a 20 cm tray (>16h) you get really nice separation of 16S and 17S.
Make 1 L of 5xSSC, 10 mM NaOH. This is your transfer buffer.
Take down the gel. Trim away excess agarose and notch a corner to help with orientation
Set up the transfer. You will need two oblong pyrex baking-type dishes, or similar. Fill one with transfer buffer. Set up your stack in the other dish:
Make a several-inch thick stack of paper towels in one dish, at least as large as the gel
Cut several pieces of Whatman filter paper a little larger than the gel. Stack these on top of the filter paper. Wet the top piece with transfer buffer.
Cut the membrane to the correct size and notch it to match your gel. Wet the membrane with transfer buffer. Place it carefully on top of your stack. Roll out any bubbles.
Puddle a few mLs of transfer buffer on top of the membrane. This helps prevent bubbles
Carefully transfer the gel to the membrane, aligning notches. Roll out any bubbles. Make sure the gel does not overhang the membrane, as this can cause your transfer to “short-circuit”.
Cut several more pieces of filter paper slightly larger than the gell. Wet them with transfer buffer. Lay them carefully on top of the gel. Roll out bubbles.
Set your dish of transfer buffer on a platform raising it a little higher than your transfer stack. Cut two or three pieces of filter paper about as tall as the gel and wide enough to span the distance to your buffer dish. These are your wicks. Wet them with buffer to prime the flow. Place one end of your wicks on your stack and the other end in your buffer dish.
Check for “short-circuits”–any place buffer can pass to your paper towel stack without going through the gel and the membrane. Block any potential short-circuits with Parafilm.
Transfer 1-2 hours. Because this buffer system is very alkaline it is not recommended to transfer overnight.
Meanwhile, make up hybridization buffer, or warm commercial buffer if you are using that. For an excellent probe I find home-made hyb buffer is quite acceptable, but for low abundance targets or less ideal probes the Ambion hyb buffers are really far superior.
Warm the hyb oven to 42°C
Disassemble transfer. Note any bubble that may be problematic
Rinse the membrane with 2x SSC
Let the membrane air-dry
Cross-link in a Stratalinker. Auto-crosslink works fine.
Place the membrane in a hyb bottle and add hyb buffer. Pre-hyb by rolling for an hour. It does not hurt to go longer
Meanwhile, label your probe. I probe with 60 pmol of oligo 323. Assemble in order:
6 µL 10 µM oligo
2 µL 10x PNK buffer
10 µL Y-32P-ATP
2 µL PNK
Incubate 30 min at 37°C, then move to 75°C for 5 min to heat-kill PNK. Dilute to 40 µL and clean up on a TE-10 column to remove free label.
At the end of the pre-hyb period add the labeled probe to the hyb bottle. Be sure to pipet it directly into the hyb buffer, and don’t let it spatter on the membrane.
Hybridize by rolling overnight at 42°C
Wash day! Use about 50 mL per wash. Dispose of radioactive waste properly!
Make sure you have
100 mL 2xSSC/0.1% SDS
200 mL 0.2xSSC/0.1% SDS
100 mL 0.1xSSC/0.1% SDS
Pre-warm 100 mL 0.2xSSC/0.1% SDS and 100 mL 100 mL 0.1xSSC/0.1% SDS to 42°C
Pour off hyb buffer. Take care, it will be VERY hot!
For all washes, carefully pour off hot buffer into radioactive waste. The first washes will be VERY hot. Pour on fresh buffer and roll in hyb oven.
Wash 2×5 min with 2xSSC/0.1% SDS at room temp
Wash 2×5 min with 0.2xSSC/0.1% SDS at room temp
Wash 2×15 min with 0.2xSSC/0.1% SDS at 42°C
Wash 2×15 min with 0.1xSSC/0.1% SDS at 42°C
During the last washes erase a small phosphoimager screen
Place membrane on a sheet of filter paper. Wrap in plastic wrap.
Expose to a screen several hours to overnight
Scan and enjoy!