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Preparation of Mouse Tail DNA for Dot Blots or PCR
These procedures were originally devised in Richard Palmiter's lab for use with tail dots (DNA spotted onto a nitrocellulose filter and probed for a transgene; quicker than doing southerns), and subsequently modified for PCR preps.
- Cut off tail tip (2-5 mm for PCR; 5-10 mm for dots), transfer to a pre-labeled tube, and store at room temperature or 4 oC until all the tails are ready (as long as 4 hrs at room temp.). If you remove 5 mm or less, you can use microfuge tubes, else use 15 ml snap-cap tubes. Typically, 2-3 mm is sufficient for PCR plus one Southern blot lane if you later need that. Some people who take off more than 5 mm of tail will sometimes do so with the mouse under light ether anesthesia, cauterizing the wound immediately afterwards.
- Add Tail Solubilization Buffer (TSB) to tubes. Use 2 ml for 1 cm fragments, 1 ml for 0.5-1.0 cm fragments, and 0.5 ml for fragments smaller than 0.5 cm.
- Incubate tubes for about 4 hrs at 45 oC to digest tail. You can use anything from overnight at room temp. to a couple hrs at 55o and it seems to work similarly. If the tail doesn't completely digest, add another 30% volume of fresh TSB and continue the digestion.
- Allow the tubes to cool to room temperature or less. I often put them at 4 oC overnight. The tails are stable this way indefinitely.
- Add 400 ul Tail Salts per 1 ml of TSB to each tube (i.e. 200 ul Tail Salts to 500 ul TSB), and vortex VERY WELL (5-10 seconds). The salts precipitate much of the protein and the potassium precipitates the SDS.
- Incubation of 30 minutes at 4 oC is sufficient if the tail solutions were already cold, else let them go longer (1-4 hours) at 4 oC.
- Centrifuge the tubes 10 minutes at maximum speed in a microfuge or 10 Krpm in an HB-4 preparative centrifuge rotor. Samples can be stored several weeks before the pellet resuspends itself, and are stable indefinitely.
- Remove some supernatant into a separate microfuge tube for precipitation with 2 volumes of ethanol. I usually use 20-80 ul of supernatant for PCR or 300 ul for tail dots, and let the DNA precipitate at least 4 hrs at -20 oC. Pellet the DNA in a microfuge at maximum speed for 5 (up to 15) minutes.
- Resuspending the DNA for PCR or tail dots:
- For PCR, remove supernatant, wash the pellets once with 80% ethanol, carefully remove all traces of wash, and allow to dry. Resuspend the pellets in 50 ul 1 mM Tris (8.0)/ 0.1 mM EDTA, and use 4 ul DNA and a 16 ul mix of the remaining components for PCR amplification (note: I've had PCR reactions fail from adding too much DNA to the reaction more often than from adding too little).
- For tail dots,
- Wash the pellets once with 80% ethanol and allow to dry. Resuspend the pellets in 12 ul DNA Denaturing Solution (or as little as 7 ul).
- Place the tubes in boiling water for 3 min (or until condensation forms on the roof of the tube).
- Bring down the condensation with a momentary microfuge spin.
- Dot 5 ul of DNA solution onto a nitrocellulose sheet marked with a 1 cm grid with parafilm or plastic wrap (not filter paper!) under the nitrocellulose. To avoid having the solution bead up on top of the paper, rinse the pipette tip once with 95% ethanol before taking up the DNA solution so that a little residual ethanol mixes in as the solution is dotted down.
- Let the dots air dry, rinse the filter in 2x SSC and then crosslink the DNA to the paper using your favorite technique. The filters are then ready for prehybridization, etc.
All solutions stored at room temperature, stable indefinitely unless otherwise noted.
10x SET: 10% SDS, 50 mM EDTA, 100 mM Tris (8.0)
Tail Solubilization Buffer (TSB): 1x SET, 100 mM NaCl, 200 ug/ml
FRESH proteinase K (enzyme added the same day)
Tail Salts: 4.21 M NaCl, 0.63 M KCl, 10 mM Tris (pH 8.0).
These are empirically optimized salt concentrations worked out in Richard Palmiter's lab. Read the pH AFTER adding all the components, since high salt concentrations change the pH dramatically. An acidic solution will cause slow degradation of the DNA during storage. If this happens, it is sometimes still suitable for Southern blotting, but rarely for PCR.
DNA Denaturing Solution: 2M NaCl, 100 mM NaOH.
Not more than two weeks old.
20x SSC: 3 M NaCl, 0.3 M Na-citrate
Eric Mercer - last modified 5/1/94 1