5.1@ DNA gel-blot analysis
Genomic DNA
is digested with one or more restriction enzymes, and the resulting fragments
are separated according to size by electrophoresis through an agarose gel. The
DNA is then denatured in situ and transferred from the gel to a solid support
(usually nylon membrane). @The relative
positions of the DNA fragments are preserved during their transfer to the
filter. @The DNA attached to the filter
is hybridized to radiolabeled or enzyme-labeled DNA, and autoradiography or
chemi-illumination is used to locate the positions of bands complementary to
the probe. @
1)
Preparation of the DNA-blotted membrane
E 2.0 µg genomic DNA
E Appropriate restriction
enzyme
E Ethanol
E 3 M CH3COONa pH 5.2
E TAE
E SeaKem GTG Agarose
E 0.25 N HCl
E Denaturing solution (0.4
M NaOH, 1.5 M NaCl)@
E Alkaline transfer buffer
(8 mM NaOH, 1.5 M NaCl)
E 2x SSC
E Hybond N+ (Amersham)
E Whatmann 3MM paper
E Paper towel
E Electrophoresis
apparatus (Nippon eido NB1011: 140 mm x 150 mm gel, BIO craft BE520: 135mm x
150 mm gel)
E Power supply
E Oven
1. Digest 2
µg of genomic DNA with an appropriate restriction enzyme.@ We usually use 15-20 units per µg DNA
and incubate the solution at 37˚C overnight.
2. Purify the
digested DNA with phenol extraction and then precipitate with ethanol.
3. Add 15
µl of TE keep a few minutes on bench, and completely dissolve by
vortexing.@ Be careful not to miss DNA
pellet which easily stick to inner tube wall during voltexing.@ Add 3 µf of 6x dye and mix.
4. Apply each
sample and marker to agarose-gel (0.7% gel) and perform electrophoresis to
separate the DNA fragments(CV = 20-25 V for 12-16 hrs).
5. After
electrophoresis, stain the gel with EtBr.@
Take a photo.
6. For
depurination, incubate the gel in 0.25 N HCl for 10 min.@ Incubation times should be limited to less
than 20 min.
7. For
denaturation, incubate the gel in denaturing solution for 1 hr.
8. After
denaturation, transfer the DNA to a nylon membrane Hybond N+ (Amersham Biotech)
by a downward method (Chomczynski (1992) Anal. Biochem. 201:134-139)
9. Rince the
membrane with 2x SSC.@
10. Dry at 80˚C
for 30-60 min.
[Key
points]
E BglII, EcoRI, EcoRV, EcoT22I, HincII, HindIII, and PstI work well
for full digestion of P. patens genomic DNA.
2)
Southern hybridization using non-RI system.
Please refer to a manual of AlkPhos direct (Amersham
Biotech: RPN3690).@ In this section, a
protocol for hybridization and detection is described.
E hybridization buffer
(refer to the manual)
E Primary wash buffer
(refer to the manual)
E Secondary wash buffer
(refer to the manual)
E CDPstar (Amersham)
1. Pre-heat
buffer and hybridization bottles at 55˚C.
2. Place a
membrane in a bottle and add 10-15 ml of hybridization buffer in the bottle.
3. Incubate at
55˚C for at least 20 min.
4. Add probe
solution in the bottle.
5. Incubate at
55˚C overnight (14-16 hrs).
6. After
hybridization, wash the membrane in first wash buffer at 60˚C for 10 min x
2.
7. Wash the
membrane in secondary wash buffer for 5 min x 2.
8. Drop 1-2 ml
of CDPstar on the membrane and incubate for 5 min.
9. Discard
excess CDPstar from the membrane, and wrap it with a Saran wrap.
10. Expose the
membrane to X-ray film for 1 hrs.
@
2)
Southern hybridization using RI-labeled probes
[Introduction]
This protocol
is described according to Church and Gilbert (1981).
1. Probe
preparation
[Procedure]
1) Add 12.5 ng
template DNA andH2O to a final volume of 5.5 µL to a microfuge tube.
2) Denature the
DNA by heating in a boiling bath or heat block and chilling quickly in an ice
bath.
3) Add 2
µL of High Prime solution and 2.5 µL of 32P-dCTP solution to
denatured DNA solution.
4) Incubate for
10 min at 37˚C.
5) Stop the
reaction by adding 2 µL of 0.2 M EDTA (pH8.0) and/or by heating to
65˚C for 10 min.
6) De-salt the
labeled probe by applying to Sephadex G50.
[Solution
required]
E Template DNA (>25 ng)
E High Prime
DNA Labeling kit (Roche: cat.no. 1 585 592)
E [a-P32] dCTP
(3000Ci/mmol)
E TE (10 mM
Tris-Hcl(pH8.0), 1 mM EDTA)
E Sephadex G50
E Heat block
E Incubator (37˚C)
2. Hybridization,
wash and detection
[Procedure]
1) Put a
membrane in a hybridization bottle.
2) Add 5~10 ml
of hybridization buffer in the bottle.
3) Pre-hybridize
the membrane in Church Buffer for 30 min at 65˚C.
4) If the probe
is double-stranded, boil 100 µl of probe for 5 min to denature the DNA
and immediate transfer it to an ice bath to cool for 5 min.
5) Add the
denatured probe to the pre-hybridization buffer, being careful not to add the
probe directly onto the membrane.
6) Hybridize
overnight at 65˚C.
7) Remove the
hybridization solution and rinse the membrane in wash buffer.
8) Transfer the
membrane to 300 ml wash buffer. Incubate for 20 min at 65˚C. Replace with
fresh wash buffer and incubate for 20 min at 65˚C.
9) Wrap the
moist (but not dripping wet) membrane in saran wrap. Do not let the membrane
dry out if it is to be either reprobed or washed further later.
10) Place x-ray
cassette. Cover the membrane with a sheet of x-ray film and one intensifying
screen. Store at -70 C overnight.
11) Develop the
x-ray film and label for marker and for each lane. @Store the wrapped membranes at –20˚C.
[Solution
required]
E 1 M ChurchNaPi buffer (0.5M NaH2PO4, adjusted
to pH7.2 with H3PO4)
E Hybridization buffer
|
Stock
solution |
/50
ml |
Final
conc. |
|
1M
Church NaPi buffer |
25
ml |
0.5
M |
|
0.5
M EDTA pH8.0 |
100
µl |
1
mM |
|
SDS
(powder) |
3.5
g |
7%
(w/v) |
|
H2O |
Up
to 50 mL |
|
EWash solution
|
Stock
solution |
/1000
ml |
Final
conc. |
|
1M
Church NaPi buffer |
40
ml |
40
mM |
|
SDS
(powder) |
10
g |
10%
(w/v) |
|
H2O |
960
mL |
|
E Hybridization oven
E Hybridization bottle
E Heat block
E Sandwich box
E Water bath
[References]
(1) Church
and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81: 1991-1995.