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.<\/p>\n
You will need a fairly large-format agarose gel. We use a tray about 20 cm long. A mini-gel will not work.<\/p>\n
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.<\/p>\n
Make 1.5 L of 10 mM sodium phosphate buffer, pH 7.0
\nPour 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.
\nMake up your samples. For total bacterial RNA, prepare ~2 ?g samples. You can use much less RNA for pure 16S or 17S control lanes.
\nTo make up samples
\n5.4 \u00b5L RNA
\n5.4 \u00b5L glyoxal
\n16 \u00b5L DMSO
\n3 \u00b5L 100 mM sodium phosphate buffer
\nIncubate at 50\u00b0C for 1h
\nDuring 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\u00b0 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.
\nAt the end of the 1 hour incubation chill samples on ice
\nAdd 4 \u00b5L glyoxal loading buffer. Vortex and spin down
\nLoad onto gel.
\nRun 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.<\/p>\n
Make 1 L of 5xSSC, 10 mM NaOH. This is your transfer buffer.
\nTake down the gel. Trim away excess agarose and notch a corner to help with orientation
\nSet 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:
\nMake a several-inch thick stack of paper towels in one dish, at least as large as the gel
\nCut 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.
\nCut 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.
\nPuddle a few mLs of transfer buffer on top of the membrane. This helps prevent bubbles
\nCarefully 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”.
\nCut 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.
\nSet 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.
\nCheck 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.
\nTransfer 1-2 hours. Because this buffer system is very alkaline it is not recommended to transfer overnight.
\nMeanwhile, 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.
\nWarm the hyb oven to 42\u00b0C
\nDisassemble transfer. Note any bubble that may be problematic
\nRinse the membrane with 2x SSC
\nLet the membrane air-dry
\nCross-link in a Stratalinker. Auto-crosslink works fine.
\nPlace the membrane in a hyb bottle and add hyb buffer. Pre-hyb by rolling for an hour. It does not hurt to go longer
\nMeanwhile, label your probe. I probe with 60 pmol of oligo 323. Assemble in order:
\n6 \u00b5L 10 \u00b5M oligo
\n2 \u00b5L 10x PNK buffer
\n10 \u00b5L Y-32P-ATP
\n2 \u00b5L PNK
\nIncubate 30 min at 37\u00b0C, then move to 75\u00b0C for 5 min to heat-kill PNK. Dilute to 40 \u00b5L and clean up on a TE-10 column to remove free label.
\nAt 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.
\nHybridize by rolling overnight at 42\u00b0C<\/p>\n
Wash day! Use about 50 mL per wash. Dispose of radioactive waste properly!<\/p>\n
Make sure you have
\n100 mL 2xSSC\/0.1% SDS
\n200 mL 0.2xSSC\/0.1% SDS
\n100 mL 0.1xSSC\/0.1% SDS
\nPre-warm 100 mL 0.2xSSC\/0.1% SDS and 100 mL 100 mL 0.1xSSC\/0.1% SDS to 42\u00b0C
\nPour off hyb buffer. Take care, it will be VERY hot!
\nFor 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.
\nWash 2×5 min with 2xSSC\/0.1% SDS at room temp
\nWash 2×5 min with 0.2xSSC\/0.1% SDS at room temp
\nWash 2×15 min with 0.2xSSC\/0.1% SDS at 42\u00b0C
\nWash 2×15 min with 0.1xSSC\/0.1% SDS at 42\u00b0C
\nDuring the last washes erase a small phosphoimager screen
\nPlace membrane on a sheet of filter paper. Wrap in plastic wrap.
\nExpose to a screen several hours to overnight
\nScan and enjoy!<\/p>\n","protected":false},"excerpt":{"rendered":"
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. […]<\/p>\n","protected":false},"author":40,"featured_media":0,"parent":32,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-62","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/pages\/62","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/users\/40"}],"replies":[{"embeddable":true,"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/comments?post=62"}],"version-history":[{"count":1,"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/pages\/62\/revisions"}],"predecessor-version":[{"id":63,"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/pages\/62\/revisions\/63"}],"up":[{"embeddable":true,"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/pages\/32"}],"wp:attachment":[{"href":"https:\/\/sites.krieger.jhu.edu\/woodson\/wp-json\/wp\/v2\/media?parent=62"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}