1. Digest good-quality genomic or plasmid DNA to completion with restriction enzymes. Use AT LEAST 1 ug  of genomic DNA per lane; 10 micrograms per lane is ideal.  If more than one DNA sample is to be compared, you should load equal quantities of each. Digestion should proceed for 4 hours, and usually overnight.

2.  Make a 0.7% agarose gel with TAE buffer.  Add loading dye to digests (1 microliter of dye per 5 microliters of sample). Load samples into wells, and electrophorese until the dye passes off the end of the gel. For a 13 x20 gel 18 hs at 30V. Running the gel slowly results in thinner, sharper bands on the Southern).

 

1. 3. Stain gel with ethidium bromide for 30 minutes. Unstain 10 minutes and photograph. Minimize exposure of the gel to UV light.

4.   The stained gel should be placed in a dish, and shaken gently in the following series of solutions for the specified times (see end of protocol for recipes):

 

1. Depurination solution: 10 minutes
2. Rinse briefly in sterile distilled water
3. Denaturation solution: 30 minutes
4. Rinse briefly in sterile distilled water.
5. Neutralizing solution: 2 X 15 minutes
6. The dry filter can be labeled with a soft lead pencil. Presoak the labeled nylon filter membrane for about 5 minutes in water, then transfer to the Neutralizing solution (membrane should be handled at the edges only with clean forceps or gloved hands).
7. Place a glass plate. Place the gel on top of it. Place the membrane on top of the gel. Avoid trapping any air bubbles between the gel and the membrane.
8. Note: Air bubbles block the transfer of nucleic acid to the membrane.  They may be removed at any stage by rolling a clean pipette or glass rod over the surface.
9. Note: Do not attempt to move the membrane once it has touched the gel surface.

 

1. Place three sheets of wet Hybond Blotting paper cut to size and saturated in transfer buffer, on top of the membrane.  Avoid trapping any air bubbles. Place a stack of absorbent towels on top of the Hybond Blotting paper at least 5 cm high.Finally, place a glass plate and a weight on top of the paper stack.  Allow the transfer to proceed overnight.  The weight should not exceed 750g for a 20 x 20 cm gel.
2. Note: Small fragments (0.5-1.5 Kb) are rapidly transferred upward in a few hours; larger fragments (> 10Kb) require at least overnight transfer.  The efficiency of transfer of these large fragments can be improved by depurination.

 

 

Place a plate of glass on top:

Glass plate

gel

membrane

wet blotting paper

paper towels (3-4 cm)

Glass Plate

 

1. Leave the blot for at least 3 hours overnight, is better.
2. Disassemble the blotter stack.
3. After blotting, carefully dismantle the transfer apparatus.  Before separating the gel and membrane, mark the membrane to allow identification of the tracks with a pencil or chinagraph pen. Discard blotting papers in the regular trash, and the gel in the ethidium bromide waste container.
4. Note: Rinsing the membrane following transfer is not advised.  Extensive experimentation at Amersham Biosciences has shown that rinsing the membrane before fixation produces blots of variable quality because nucleic acid is removed from the membrane during this step.

 

1. Fix the nucleic acid to the membrane by using an optimized UV crosslinking procedure.

 

1. Place the blot inside the Stratalinker (3 floor) on a clean paper towel. Press the auto-crosslink button, then the start button. When the cross-linking is complete dry the blot on the lab bench and seal it in a heat-seal bag until use.

 

1. Blots may be used immediately. Blots must be thoroughly dried if storage is required.
2. Note: Blots may be rinsed in 2x SSC before storage or hybridization.  Blots should be stored wrapped in SaranWrap desiccated at room temperature under vacuum.

 

 

 

 

Recipes for Part I

 

·      Depurination solution

11 ml HCL

989 ml Distilled Water

Mix. Store at room temperature for up to 1 month.

 

·      Denaturation buffer

87.66g NaCl

20g NaOH

Add approximately 800ml of distilled water.  Mix to dissolve.  Make up to a final volume of 1000ml.  Store at room temperature for up to 3 months.

 

·      Neutralization buffer

87.66g NaCl

60.5 Trizma base

Add approximately 800ml of distilled water.  Mix to dissolve.  Adjust to pH 7.5 with concentrated hydrochloric acid.  Make up to a final volume of 1000ml.  Store at room temperature for up to 3 months.  (Will take a few ml of acid)

 

·      Nucleic acid transfer buffer (20x SSC)

88.23g Tri-sodium citrate

175.32g NaCl

Add approximately 800ml of distilled water.  Mix to dissolve.  Check the pH is 7-8.  Make up to a final volume of 1000ml.  Store at room temperature for up to 3 months.

 

 

 

Southern Blot Part 2:  hybridization and labeling the probe

 

 

 

Hybridization

 

Turn on the oven and set to 50 C. Place empty bottles in the holder to pre-warm.

 

Pre-wet the blot in a suitable dish, first in water then in an appropriate buffer.  Ensure that the nucleic acid side is uppermost.  Roll the blot along its length in such a way as to minimize overlap in the tube.  Place inside the hybridization tube.

 

Note: If there is significant overlap of the blot use a nylon mesh should be considered.  The mesh achieves separation of the blot layers allowing better probe access to these areas.  It is strongly advised that hybridization volume should be increased (70-125 ml/cm²).

 

The nylon mesh should be at least 0.5 cm larger than the blot.  Place the mesh in the pre-wetting solution before the blot, in subsequence manipulation treat as ‘one’.   The nylon mesh may be reused after washing in 10% (w/v) SDS and extensive rinsing in distilled water.

 

Preheat hybridization solution Ultrahyb-(Ambion,)at 68C until any precipitate has dissolved.  10 ml for each for each blot and place in the hybridization oven to warm up (If the blot is big 20 ml will work better).  Unroll the blot by rotation the tube in the opposite e direction to the ‘rolled’ blot.

 

 

Pre-hybridizate in the solution and place in the rotisserie to pre-hyb the blot for at least 30 minutes (blot can pre-hyb longer if necessary). Then replace the solution with new hybridization solution for another 30 minutes. at 44C.

 

Note: High backgrounds will result if sub optimum volumes are used for the membrane and hybridization conditions.

 

Note: It is important not to allow air to become trapped between the inner surface of the tune and the membrane.  This can cause areas of no signal or background following hybidization.

 

1. Hybridize overnight at the required hybidization temperature.

 

Prepare the stringency wash solutions.  The wash solutions should be used in excess.  Use a volume that occupies 33-50% of the tube.

 

Low stringency wash; 2x SSC, 0.1% (w/v) SDS

Medium stringency wash:

1x SSC, 0.1% (w/v) SDS

High stringency wash:

0.1x SSC, 0.1% (w/v) SDS

 

Note: Hybidization temperatures may vary with the probe.  Lower temperatures achieve lower stringency.  The temperature hybridization used will depend on the degree of homology between the probe and the target.  65-68°C is suitable for most long probes (> 100 bases).  With short/oligo probes (< 50 bases) hybidization temperatures are usually defined as Tm-5°C. 

 

Tm (melting temperature) = (4 x number of G+C bases) + (2 x number of A+T bases) (14)

 

Hybridization time can also vary.  Short hybridization times may be suitable for high target applications.

1.  
2.  
3. After the hybridization wash the blot as follows:

a.    rinse briefly in 2x SSC, 0.1% (w/v) SDS

b.    wash twice, 5 minutes each in 2x SSC, 0.1% (w/v) SDS

c.     wash twice, 10 minutes each 1x SSC, 0.1% (w/v) SDS

d.    wash four times, 5 minutes each in 0.1x SSC, 0.1% (w/v) SDS

 

Note: Washing in boxes is much more effective and is recommended if feasible.  The inefficiency of washing in tubes may be overcome by increasing the number of stringency washes while maintaining the same total wash time.  

 

1. Remove the blot form the last stringency wash, drain and wrap in SaranWrap and expose to X-ray film, for example Hyprfilm MP.  Keep the blot moist if it is to be reprobed. 

 

Note: The use of SaranWrap with ³⁵ labeled probes will significantly increase exposure time.  In this case the blot should be air dried before autoradiography, if reprobing is not required.

 

 

Labeling  of  the probe

 

Remember to take out of the freezer  dCTP (P³² ).

 

Label about 30ng of the DNA probe/ reaction.   We use the TAKARA kit instructions. After step 5) and before step 6) clean the probe.

 

To clean the probe: Prepare cleaning column snap or cut bottom of the column and place it into a 2 ml collection tube and centrifuge at 3000´g for 3min

 

apply the labeled probe to the column and place the column in a fresh 1.5ml tube centrifuge at 3000´g for 3min to remove the unlabeled probe. Discard the filter in the radioactive waste container (this contains the unincorporated 32P-dCTP).  Use probe immediately or store at  in the freezer at -20°C. Check microfuge with Geiger counter after spin.

 

denature the probe at 100°C for 5min, then  transfer quickly to ice for 5min. Measure with the Geiger counter at 100x.

 

Add the probe to the hybridization bottle. Pour some the hyb sol from the bottle to a 50ml tube and add the label probe mix and pour back in hybridization bottle.

 

Return the bottle to the oven and incubate for at least 16 hours (overnight) at 42C.  Blot can hybridize longer if necessary.  The rotisserie should be set at a speed of 7.

 

Pour off the hybridization solution. This can be placed in a plastic tube, labeled with the date and the name of the probe, and stored at -20 C to be used again (see above).

 

Complete the following washes.  Both washes should be performed at 42 degrees in the hybridization oven.  Preheat the wash solutions for at least one hour in the hyb oven.

 

 

Solution 1:  2 X 10 minutes, at 42 C

            solution 1: 100 ml of 2X SSC/0.1% SDS

                        10 ml                         20X SSC

                        1 ml                        10% SDS

                        89 ml                         water

 

Solution 2: 2 X 15 minutes. At

            solution 2: 100 ml of 0.1X SSC/0.1% SDS (pre-warm to 65 C for a high stringency blot).

                        0.5 ml             20X SSC

                        1 ml                        10% SDS

                        98.5 ml             water

For a lower stringency blot, the temperature of this second rinse can be lowered.  The exact temperature must be determined empirically.

 

 

Probe stripping

 

1.    Wash the blot 2 X 15 min in probe stripping solution (0.2 M NaOH, 0.1% SDS) at 37 degrees.  Preheat both the glass dish and the probe striping solution to 37 degrees before proceeding.

 

2.    rinse 2-3 X 10 min at room temp in 2X SSC.

 

Note:  The DIG system uses and alkali labile DIG-dUTP conjugate.  I have used this protocol to strip and reprobe genomic DNA blots 4-5X with no detectable signal from the previous probe.

 

 

1 M sodium phosphate pH 6.8

138g NaH2PO4 € H2O
268g Na2HPO4 € 7 H2O

Adjust to 2 liters with DD H2O.
Autoclave.