Southern hybridization
KIT and Reagent
PCR DIG probe synthesis KIT
(roche) #11636090910
DIG Easy Hyb
(roche) #1603558
CDP-Star
(roche) #12041677001
Anti-Digoxigenin-AP, Fab fragment
(roche) #1093274
Blocking reagent
(roche) #1096176
2 x Wash buffer
2 x SSC
0. 1% SDS
0.5 x Wash buffer
0.5 x SSC
0. 1% SDS
1 x MAB
0. 1M Maleic acid
0. 15M NaCl
MABT
1 x MAB
0. 3% Tween
Blocking solution
1 x MAB
1% Blocking reagent
Antibody solution
Anti-Digoxigenin-AP (1:10,000) in blocking solution
Detection buffer
0. 1M Tris-HCl(pH9.5)
0.1M NaCl
Dig probe synthesis
DIG (+)
DIG (-)
10 x Buffer (3)
5
5
DIG mix (2)
5
-
d NTP mix (4)
-
5
primer F (20uM)
2.5
2.5
primer R (20uM)
2.5
2.5
Enzyme mix (1)
0.75
0.75
1
1
H2O
xx
xx
total
50 ul
50 ul
template DNA
PCR reaction
95C 2min
95C 30sec
55C 30sec
30 cycles
72C 40sec
72C 7min
Optimize PCR condition for your sample.
After the reaction, rune 5ul of each sample on a gel.
The DIG labeled PCR product should be larger band than the unlabeled products. The
amount of labeled probe is equal or less than the amount of unlabeled probe. Then, the
reaction is OK
Use 0.5 - 4 ul / ml hybridization solution.
Hybridization
※ This protocol is using 225 cm2 (15 cm×15 cm) size membrane
1.
Place the membrane into a hybridization bag, add pre-warmed 22.5 ml DIG Easy
Hyb to the bag (10ml / 100 cm2)
2.
Heat seal closely around the membrane, while removing air bubbles.
3.
Incubate the membrane for at least 30 minutes at 42C (Prehybridization)
4.
Prepare the hybridization solution
4.1
Mix 7ul of DIG-labeled probe and 50ul of H2O, 100C for 5 minutes to denature
the probe.
4.2
Chill the probe quickly in the ice.
4.3
Immediately add the denatured probe to a tube containing pre-warmed 7.7 ml
DIG Easy Hyb (3.5 ml / 100 cm2)
5.
Pour out the prehybridization buffer, immediately add hybridization containing
DIG-labeled probe to the bag, heat seal the bag while removing air bubbles.
6.
Incubate the membrane with probe over night (Hybridization).
Wash and Detection
1.
Prewarmed 0.5 x SSC at 65C.
2.
Add 200 ml 2×Wash buffer to a plastic tray.
3.
Immediately place the membrane in a plastic tray with 2×Wash buffer.
4.
Incubate the tray twice at room temperature for 5 minutes with shaking.
5.
Pour off 2×Wash buffer, immediately add the preheated 100 ml 0.5×Wash Buffer
to the tray containing the membrane.
6.
Incubate the membrane twice at 65C for 15 min with shaking
7.
Transfer the membrane to another plastic container containing 100ml MABT.
8.
Incubate for 2min at room temperature with shaking.
9.
Discard MABT, add 100 ml Blocking Solution to tray
10. Incubate for 30min at room temperature with shaking (Blocking).(This blocking
step can last up to 3 hours without affecting result)
11. Discard the Blocking Solution, add 25 ml Antibody Solution to the tray.
12. Incubate the membrane for 30min at room temperature with shaking
13. Discard the Antibody Solution, wash the membrane twice (2 x 15 min) with 100 ml
MABT.
14. Equilibrate membrane 3 min in 20 ml Detection Buffer.
15. Place the membrane inside hybridization bag, apply 2-3 ml CDP-Star (1ml / 100
cm2), dropwise, over the surface of the blot until the entire surface is evenly soaked.
16. As you are applying the substrate, immediately cover the membrane while remove
air bubbles.
17. Incubate membrane for 5 min at room temperature.
18. Squeeze excess liquid out and seal the bag
19. Expose the sealed membrane 10~20 min, then detect with LAS-1000 (Rm 315) (Put
the membrane on the third shelf from the top)