Southern Blots with Alkaline Transfer

Procedure

1. Digestions

  • 10-15 uL Genomic DNA (from ES prep)
  • 2-3 uL Enzyme
  • 4 uL 10X Enzyme Buffer
  • 0.4 uL 100X BSA
  • 1.6 uL 100 mM Spermidine
  • X uL Water (DNase free)
  • 40 uL Total

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.

2. The Gel

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.

3. After the gel

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.

4. Transfer

The sandwich - in a big tray set up the following from the bottom up....

  1. Big sponges or paper towels (2-3 inches in height) in 0.4 N NaOH.
    Sponges/towels must be larger than the gel.
  2. 2 sheets of Whatman filter paper cut slightly larger than gel size.
  3. Gel, open wells face down. (Now the cut corner is on the top right.)
  4. Hybond-N+ membrane (GE Biosciences) (pre-soaked in 0.4 N NaOH, top right corner removed for orientation).
    Line up top of the membrane to the wells of the gel, with cut corners in the same quadrant. For further orientation, you can mark the membrane with pencil or VWR marker. Do not move the membrane around once settled on gel.
  5. Sheets of Parafilm to surround gel like a frame and prevent wicking
  6. 2 sheets of Whatman filter paper cut slightly larger than gel size.
  7. Big stack of paper towels. 4-5 inches is good.
  8. Weight - a smooth piece of plastic with a heavy book (or 2)on top works well.
    Add plenty of 0.4 N NaOH to the tray. Let go for at least 4-6 hours, though overnight is great.

5. Rapid Hybridization

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!

6. Hybridization

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).

7. Washing!

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.

8. Develop!

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!

Recipes

For 1 Liter of 20xSSPE

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

For 1 Liter of 50xTAE

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

Neutralization Buffer

 AmountFinal Concentration
20X SSPE 10 mL 2Xz
1.0 M Tris pH 7.5 20 mL 0.2 M
H2O 70 mL  
  100mL Total

FBI (Pre-Hybridization and Hybridization Buffer)

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).