Southern Blotting
Genomic DNA isolation
Use invitrogen Easy DNA isolation kit
- Grow bacteria for 6~8 hr
- Centrifuge 1.5 ml of broth culture
- Resuspend the cells with 200 l of TE plus 6 l of Lysozyme (100 mg/ml)
- Incubate for 1 hr at 37C
- Follow the steps described in the kit manual.
- Quanitify the concentration of DNA
* DNA conc.: 1~2 g/l in 100 l
* Ratio 260/280 >1.9
* DNA is highly soluble.
- Confirm DNA integrity by running electrophoresis of 100~200 ng of DNA on
1% Agarose gel at a constant V (5~10V/cm gel length)
Preparation of Agarose Gel
50X TAE
Working solution
Concentrated stock
1X: 40 mM Tris-acetate
50X: 242 g Tris base
1 mM EDTA
57.1 ml glacial acetic acid
100 ml 0.5M EDTA (pH 8.0)
Prepare 100 ml agarose gel sol. (in 1X TAE) of desired percent and
dissolve it in a microwave oven and add 5 l of EtBr (10mg/ml)
Restriction Enzyme Digestion
- Analyze restriction digestion sites of the target sequence using Mapdraw.
- Select suitable enzymes that do not cut target DNA inside.
- Prepare master mix solution for restriction enzyme.
- Use ~15 g of genomic DNA in 30 l reaction
- Use 5 U per 1 g genomic DNA
- Be sure not to generate Star activity of some enzymes.
- Decrease the final glycerol % lower than 5%
(Enzyme sol. has 50% glycerol)
Xba I digestion of Bacillus Genomic DNA (NEB)
Reaction vol. -->
30
x
1
4
Sterild D.W.
19.7
78.8
10X Buffer 2
3
12
BSA, 10ug/ul
0.3
1.2
R.E., 20u/ul (use 5U for 1ug Target DNA)
3
12
Total minus DNA
26
104
Target DNA in each tube (3 ug/ul)
4
(~7 ug (15 ul) for 1 well of 24x comb for Southern blot)
Aliquot
11
216.7
33
3.3
33
286
20
394
60
6
60
520
30
591
90
9
90
780
26 ul in the microtubes, and add the Target DNA
Incubate at 37C, 4 hours
- Mix the final solution carefully and gently using P200 pipette up and down 10 times.
- PCR tubes can be used.
- Thermocycler can be used for incubation.
4x 37C 60 min, 65C 20 min, 4C 
DIG Luminescent Detection
Preparation of Stock Solutions
Dilution buffer
0.1 M Maleic acid, 0.15 M NaCl; pH 7.5
Don’t try to make 10X concentrated buffer,
it’s very difficult to dissolve Maleic acid.
Washing buffer
Dilution buffer + 0.3% (v/v) Tween 20
Detection buffer
0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5
10x concentrated buffer can be prepared.
Preparation of fresh working solutions just before use
Blocking solution
Always prepare fresh just before use. Prepare during stringency wash steps.
Dissolve 2 g of Blocking reagent in 200 ml of heated (>60C) Dilution buffer
Cool it down to ambient temperature.
Procedure (for 200 cm2 membrane)
1.
Blocking solution should be already prepared.
2.
Equilibration of the membrane (after hybridization, stringency washes) with 200 ml
Washing buffer for 1-5 min.
3.
Incubate for 1~3 hr in 200 ml Blocking solution.
4.
Incubate for 1~2 hr in 60 ml Antibody solution.
Anibody solution
Centrifuge Anti-Digoxigenin-AP for 5 min at 10,000 rpm in the original vial
prior to each use, and pipet the necessary carefully form the surface.
Dilute antibody 1:10,000 in used blocking solution in the blocking step.
2x (4.5 ul in 30 ml Blocking sol. in 50ml cornical tube) = 60 ml
5.
Wash 2 x 15 min in 200 ml Washing buffer.
6.
Equilibrate 2~5 min in 40 ml Detection buffer.
7.
Place membrane with DNA side facing up on a hybridization bag opened by cut on both
lateral margin and apply 4 ml diluted CSPD solution
CSPD
Dilute CSPD 1:100 in Detection buffer
(can be reused one to two times)
40 ul in 4 ml in detection buffer for 200 cm2 membrane
Immediately cover the membrane with the second sheet of the hybridization bag to spread
the surface evenly and without air bubbles over the membrane.
Incubate for 5 min at room temperature
8.
Squeeze out excess liquid and seal the edges of the hybridization bag
9.
Incubate the damp membrane for 5~15 min at 37C to enhance the luminescent reaction.
10. Expose to Chemi-Imager for 30~150 min
How to set the Chemi-Imager
- Place the hybridization bag on the dark surface (UV chamber box) and fix the
membrane position by putting the weights on the surrounding area of membrane.
- Remove any light filters.
- Set the magnification and focus
- Turn off any light sources within the Imager chamber
- Open the diaphragm maximally.
- Press reset button of the image software.
- Set the exposure time 30~150 min.
- During the exposure, don’t open the door of the chamber and don’t touch any sets.
- After exposure is finished, press the freeze button and save the picture.
- Press Inverse image button
- Adjust Black and White degree for optimal signals to background.
11. For reprobing, store the nylon membrane in 2x SSC or process stripping protocols.