SECTION

Southern Blot

DESCRIPTION

S o u t h e r n blotting is a technique used t o transfer DNA from its position in an

agarose gel t o a nitrocellulose (NC) filter placed directly above t h e gel. T h e DNA
is d e n a t u r e d , neutralized, and transferred in a high-salt buffer by capillary
action. T h e denatured, single-stranded DNA binds to t h e filter, is p e r m a n e n t l y
b o n d e d by baking t h e filter, a n d is later hybridized t o a radiolabeled p r o b e t o
d e t e c t hybridizing DNA species.

TIME REQUIRED

Day 1—3 hr
Day 2 — 3 hr

REAGENTS

Solution A: To m a k e 1 liter
300 ml 5 Μ NaCl
50 ml WMNaOH
650 ml H 0 2

Solution B: To m a k e 2 liters
200 ml 10 Μ ammonium acetate1

4 ml WMNaOH
1,796 ml H 0 2

NC filters, 20 x 20 c m
2

1
Acetate is now used in buffer instead of NaCl for better removal in vacuum oven due
to its higher volatility.
2
Gene Screen Plus (NEN) or nylon can be used in place of NC because they are more
durable for reuse. Method conditions will change; follow manufacturers' recommen­
dations.
62
 5-6. Southern Blot 63

METHODS
In Advance
Run electrophoretic separation of DNA on an agarose gel (Section 5-5). T a k e
p h o t o g r a p h of ethidium b r o m i d e - s t a i n e d gel (Section 20-4)

Day 1
1. P u t e a c h agarose (typically 20 x 20 cm) gel in a plastic b o x with 500 ml of
solution A at r o o m t e m p e r a t u r e . Agitate periodically by hand. After 30 min,
replace with 500 ml of fresh solution A. Continue periodic shaking for
a n o t h e r 30 min. This s t e p is designed to d e n a t u r e double-stranded DNA.
2 . To r e t u r n gel t o a m o r e neutral pH, replace liquid with 500 ml of solution B.
S h a k e periodically, a s above, for 30 min. Replace with 500 ml of fresh
solution Β a n d s h a k e periodically for 30 min.
3· P u t 500 ml of fresh solution Β in a n o t h e r plastic dish. Wet W h a t m a n 3MM
filter with solution Β to act eventually as a wick. We u s e a 50- t o 60-cm-long
piece t o r n from a 23-cm-wide roll. Place a 20 x 30-cm plastic or glass plate
a c r o s s t h e t o p of t h e dish and place wick over plate with e n d s hanging
d o w n into t h e solution. Smooth wick o n t o p of plate by rolling with a 5-ml
glass pipette t o r e m o v e t r a p p e d b u b b l e s that distort transfer (see Figure
5-3A).
4 · Carefully lift gel from dish containing solution Β in s t e p 2 by sliding a glass
p l a t e u n d e r n e a t h it for t r a n s p o r t . Cover gel w i t h a s e c o n d p l a t e . Carry gel
o v e r t o wick. Flip gel over, a n d r e m o v e t h e t o p glass p l a t e , a n d slide gel
o n t o wick. Align gel w i t h e d g e of wick a n d p l a t e . S m o o t h gel a n d r e m o v e
t r a p p e d bubbles b y rolling a glass p i p e t t e o v e r surface (see Figure 5-3B).
N o t e : Gel is n o w u p s i d e - d o w n , b e c a u s e it is b e s t t o blot from t h e b o t t o m for
s h a r p e r resolution a n d t o maintain t h e left-to-right sample orientation o n t h e
b l o t t e d NC filters.
5. P r e w e t 20 x 20 c m NC filter in water. Place filter directly over gel. S m o o t h
3

as above. 4

6· P r e w e t W h a t m a n 3MM 20 x 20 c m filter in w a t e r or solution B . Place over 3

NC filter and s m o o t h as above. Cover with a s e c o n d 3MM filter. S m o o t h a s

4

above.
7. Six s h e e t s of dry, flat p a p e r towels are placed individually over t h e 3MM
filters. They act t o start capillary action, drawing liquid u p t h r o u g h filters.
5

8. Place four additional 3-cm-thick s t a c k s of folded p a p e r t o w e l s o n t o p ( t w o

w a d s in e a c h of t w o directions).

For uniformity, reduced manipulation, and decreased waste, it is helpful to buy gel
formers, paper, and NC filter in a uniform 20 χ 20 cm size.
All smoothing is done by gently rolling a 5-ml glass pipette over the surface.
We use opened Scott 175 (brown) single-fold paper towels because they have good
absorbance, are inexpensive, and are easy to handle.
 A Β
Wick Draped Over Gel Placed
Glass Plate Over Wick

Pape
Wick

Glass
Plate Gel
(Sample
Wells Down)

Weight

Glass
is: Plate
\s\ws
Paper
Towel
Wads

6 Paper Towels
Whatman 3MM
Nitrocellulose Filter
>—Gel
Glass Plate
Wick

Buffer

JJ<- Tray
Layers of Blot
Expanded Cross Section

Figure 5.3
Preparation of a Southern or Northern blot. (A) Position of wick over
glass plate. (B) Gel is placed on wick. (C) Schematic diagram of final
layered organization of materials.
 5-6. Southern Blot 65

9. T o p with glass plate and place a light weight (approximately 250 g) on t o p

of plate (see Figure 5-3C). Allow 4 h to overnight for DNA transfer to NC.

Day 2
10. R e m o v e weight, t o p glass, and p a p e r towels. Slice off p r o t r u d i n g wick
e n d s . Wick e n d s will easily tear off along edge of b o t t o m plate.
6

1 1 . Flip over filters/NC/gel "sandwich." Remove wick, which is n o w on t o p .

Make single pinholes in c e n t e r s of all sample wells d o w n through NC t o
m a r k their position. Make additional pinholes over first well for later orien­
tation/identification. Note that the left-to-right orientation is n o w in t h e
s a m e order as w h e n samples w e r e loaded.
12. R e m o v e gel and discard. Allow a few minutes for filter t o dry. Carefully
m a r k gel n u m b e r on t o p of NC with a ballpoint pen. It is useful t o n u m b e r
all gels sequentially (Skilcraft ballpoint p e n ) and k e e p track of t h e n u m b e r ,
date, conditions, samples, and so on in a log book. Put NC filter b e t w e e n
t w o 20 x 20 c m Whatman 3MM filters. Secure on t w o sides with small
p i e c e s of masking tape.
13. Place in v a c u u m oven for 2 hr at 80°C to b a k e DNA o n t o t h e filter.
14. Store carefully ( b e t w e e n 3MM filters) at r o o m t e m p e r a t u r e until u s e d for
hybridization.

REFERENCES
Smith, G., and Summers, M., Anal. Biochem. 109:123, 1980.
Southern, E., J. Mol. Biol. 95:503, 1975.

Wear gloves for all day 2 procedures.