1% blue dextran (light blue-aquamarine)
Store buffer at 4°C (aliquots can be kept at room temp for 2 months)
3. Load the reaction. For the greatest efficiency, try to squirt it down to the top of the resin, along the side of the column.
4. Once the reaction has entered the resin, fill the column with TE-0.2% SDS.
5. The labelled DNA will elute with the blue dextran. Just before the bromophenol blue elutes, remove the column. Cap the tube.
6. On the geiger counter a "good" probe will register >2x105 cpm.
NUCLEIC ACID HYBRIDIZATION (BLOTS, COLONY LIFTS, ETC).
1. Prehybridization:
• Place the membrane in a glass hybridization tube. If you are using multiple membranes it is OK to stack them up.
• Add hybridization buffer: 0.5 M NaHPO4 pH 7.2, 7% SDS, 0.25 mM EDTA (made from 1 M, 28%, and 0.5M stock solutions). Add about 10 ml for a 10 x 20 cm membrane.
• Screw on the cap (not too tight or else it will crack!) and rotate at 65°C for 10-60 min at speed 4. Make sure you balance the tubes.
2. Hybridization:
• Remove "excess" liquid (the blot will go faster in a small volume). Generally, I leave about 2-3 mls of "free" liquid in the tube (4-5 mls for the large tubes), i.e. beyond that needed to wet the membrane.
• Boil the probe for five minutes, and then cool on ice.
• Add the probe.
• Seal the tube and rotate 12-24 hr at 65C. Maximum hybridization is generally obtained after about 16 hrs (about 3 Cot). Of course, when very strong signals are expected, shorter times can be employed.
• Make sure that the tubes rock back and forth and aren't leaking!
3. Washing:
• IMPORTANT: Never let the membrane dry out, which will fix the probe to the membrane!
• The following procedure is adequate for most applications (up to 5 blots with <250 uCi of probe). If an unusually large amount of radioactivity or blots are being washed, more washes or time may be required.
• Prepare about 1.8 liters of each wash solutions at 65C on the hot plate:
Watch the temperatures! Don't let them get too hot, esp. the final wash.
First washes: 1X SSPE, 0.2% SDS, 0.1% sodium pyrophosphate
Final wash: 0.2X SSPE, 0.2% SDS, 0.1% sodium pyrophosphate
• Pour off the probe into the liquid radioactive waste. Add 20-30 ml of 1X solution. You can dip a little beaker into the large beaker to get the 20-30 ml. Rotate about 5 minutes at speed 4 to 8.
• Repeat for a total of 3 washes. These steps remove most of the radioactivity, so that the next washes (ca. 3.5 liters) are "nonradioactive" [i.e. background counts] and can be poured down the sink.
• Using gloved fingers, carefully remove the membrane from the tube and place in the 1 X solution at 65C. Stir for 10 minutes.
• Transfer the membrane(s) to the 0.2X solution at 65C (watch the temperature!!!). Stir for 10 minutes.
• Remove the filters and carefully wrap in plastic wrap. Do not let them dry out!. Expose to Xray film using intensifying screens at -80C, or to a phosphorimager screen at room temperature.
• If the 1 X and 0.2X wash solutions in the beakers are close to background, they can be poured down the sink. Rinse out the beakers, being careful not to shock them with cold water (which might crack them): you should dump out the hot solution, wait a few minutes and then rinse out the beakers.
4. Stripping off probe:
• When done with the blot remove the probe, so that the membrane can be used again.
• Heat 0.1 % SDS, 0.1X SSPE (or SSC) to 95C on the hotplate. Turn off the heat. Add the membranes.
Incubate 20 minutes.
• Remove the membranes and blot dry. Store dry at room temperature.
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