Spermadine can be purchased from GE Biosciences, #US21760
It is always a good idea to run a control. If using plasmid DNA or BAC DNA, use 1 ng or LESS. Too much will overwhelm the signal of the real bands.
Digest 6 hours to overnight (usually ON is the easiest/best).
This is a good time to make 20xSSPE, 1M Tris pH 7.5 and TAE if you don't have them already. Recipes at the bottom of the protocol.
Pour low percentage (usually 0.6% to 0.8%) agarose gel in 1X TAE. TAE is better than TBE for resolving larger fragments. The gel should be very long (25-40cm) to give good separation of bands. Add ethidium to gel and ALLl running buffer.
Run gel 5-6 hours at 40 to 60 volts. You can also run overnight at 20 to 25 volts. Once gel is finished running, be sure to take a picture with a ruler that glows under UV!!! (Line-up zero on ruler with wells).
Cut off top, left corner of gel (near your marker) for future orientation.
Add 500 mL of 1:50 dilution of HCL (490 mL H2O, then add 10 mL HCl) to depurinate. Shake/rock gently for 15 minutes. BE CAREFUL! Lower % gels LOVE to crack.
Add 500 mL of 0.4 N NaOH to neutralize (480 mL H2O plus 20 mL 10 N NaOH).
(Add base to the water, not the other way around. Make a big batch of this!)
Shake gently 15 minutes. Watch for the dye front color to change.
The sandwich - in a big tray set up the following from the bottom up....
Neutralize blot in 100 mL neutralization buffer - 15' or so.
Dry membrane - putting it between two pieces of Whatman paper is good. This isn't important, but it helps if the membrane is a little dry to get into hybridization oven tube. Membrane can also be stored in saran wrap in a refrigerator for a while at this stage. Also, no need to crosslink! Transfer in basic solution takes care of that.
Make up FBI buffer! (also called pre-hybridization buffer at this stage) Keep it warm.
If you have FBI on your self, heat now to 55-65 °C.
Once heated and in solution, add 20 mL FBI buffer to 30 cm tube Hyb oven tube. Prehyb for at least 1 hour in 65 °C oven.
Now is a good time to make HOT probe. It is always a good idea to do this fresh!
Make Hybridization Buffer (FBI+hot Probe).
Add 1 × 106 cpm/mL of denatured probe (denature at 100 °C for 5', secure the top of tube with parafilm or cap lock) to 5-10 mL of pre-heated FBI buffer (5 × 106 to 1 × 107 cpm total).
Discard "old" prehyb buffer, add hot hyb buffer and incubate at 65°C for at least 6 hours (ON good).
All washes done at 65 °C.
Remove Hyb buffer (discard in radioactive waste).
Washes 1-3 are in 1X SPPE, 1% SDS, 30' each.
Wash 1 & 2 - just 20 mL. (in Hyb Oven tube, discard wash in Radioactive waste)
Wash 3 - 100 mL (or more). (in Tuperware, discard down drain, noting on sink's radiation log)
Wash 4 in 0.5X SPPE, 1% SDS. 30' (100 mL or more, down sink).
Wash 5 in 0.1X SPPE, 1% SDS. 30'(100 mL or more, down sink). Gently polish membrane with kimwipe after last wash to lower background; discard kimwipe in radioactive waste.
If you have tried this protocol with your probe before and gotten only a weak signal, try omitting Wash 4 and/or 5.
Mount membrane on filter paper (tape it down). Wrap in saran wrap and pop it in film cassette with intensifying screen. Store at -80°C ON (at least 15 hours) - may take longer for good exposure. Remember, the notched corner is where your ladder is!
600mL Water
175.3g NaCl sodium chloride
27.6g NaH2PO4 sodium phosphate monobasic
9.4g EDTA powder FW=372
Bring up to 800mls with H2O
Add NaOH to pH 7.4 (~27mls/liter of 10N NaOH)
Autoclave for 20 min (wet cycle with 2 inches water in bottom of tray)
Add H2O to bring final volume to 1 Liter
600mL water
242 g Tris base
57.1mls acetic acid
1/10 mole of EDTA ie 37.2g of Na2EDTA 2H2O
Bring up to 800mls with H2O
Autoclave for 20 min (wet cycle with 2 inches water in bottom of tray)
Add H2O to bring final volume to 1 Liter
Amount | Final Concentration | |
---|---|---|
20X SSPE | 10 mL | 2Xz |
1.0 M Tris pH 7.5 | 20 mL | 0.2 M |
H2O | 70 mL | |
100mL | Total |
Amount (for 1 blot) | Amount (for 1L) | Final Concentration | |
---|---|---|---|
20X SSPE | 3 mL | 75mL | 1.5X |
PEG 8000 | 4 grams | 100g | 10% |
SDS | 2.8 grams | 70g | 7% |
H2O | About 35mL | About 850mL | |
40 mL | 1 Liter | Total |
Heat 55-65 °C. Mix often (it takes a while to get in to solution).