5.@ DNA and RNA
gel-blot and RT-PCR analyses
DNA
gel blot analysis is one of the most effective methods to estimate transgenic P. patens in which transgenes are integrated
in the genome. To eliminate the risk of mistaking other effects such as genome
rearrangement and multiple-insertion for transgene effects, we recommend
selecting the transgenic lines that have simple and predictable insertion of
transgenes.
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 chemiluminescenceis used to locate
the positions of bands complementary to the probe. @
1) Preparation of the DNA-blotted
membrane
E 3 -5
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 Nylon
membrane 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 3
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. To purify
the digested DNA, precipitate with ethanol.
3. Add 17
µl of TE, keep a few minutes on bench, and completely dissolve by vortexing.@ Be careful not to miss the DNA pellet which often
sticks to the inner tube wall during vortexing.
4. (Check
1µl of digested DNA by electrophoresis)
5. Add 1.6
µl of 10x dye and mix.
6. Apply each
sample and DNA ladder marker to agarose-gel (0.7% gel) and perform
electrophoresis to separate the DNA fragments (Constant Voltage = 50 V for 30
min and 20 V for 12-16 hrs).
7. After
electrophoresis, stain the gel with EtBr and take a photo with a scale.
8. For
depurination, incubate the gel in 0.25 N HCl for 10 min at room temperature.
9. Rince with
H2O once
10. For
denaturation, incubate the gel in denaturing solution for 1 hr.
11. After
denaturation, transfer the DNA to a nylon membrane Hybond N+ (Amersham Biotech)
by a downward method (Chomczynski (1992) Anal. Biochem. 201:134-139) 1-2hr
12. Rince the
membrane with 2x SSC.@
13. Dry at
80˚C for 60 min.
[Key points]
E BglII,
EcoRI, EcoRV, EcoT22I, EcoT14I, HincII, HindIII and BamHI work well for full
digestion of P. patens genomic DNA.
2) Hybridization
(1) Using non-RI system.
Please refer to the AlkPhos direct manual (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 10ml 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 55˚C for 10 min,
twice.
7. Wash the
membrane in secondary wash buffer for 5 min, twice.
8. Drop 1-3
ml of CDPstar on the membrane and incubate for 5 min.
9. Discard
excess CDPstar from the membrane, and wrap it with Saran wrap.
10. Expose the
membrane by LAS3000 (high-mode, 2-3hr: super-mode, 1-2hr) or by X-ray film for
1 hr.
@
(2) Using
RI-labeled probes
This
protocol is that of Church and Gilbert (1981).
E Template
DNA (>25 ng)
E High Prime
DNA Labeling kit (Roche: cat.no. 1 585 592)
E [ż-32P]
dCTP (3000Ci/mmol)
E TE (10 mM Tris-HCl(pH8.0), 1 mM EDTA)
E Sephadex
G50
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 |
|
E Wash
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 Sandwich
box
E Water bath
E Heat block
E Incubator
(37˚C)
Probe preparation
1. Add 12.5
ng template DNA and H2O to a final volume of 5.5 µL in an
eppendorf tube.
2. Denature
the DNA by heating in a boiling water 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 and remove unincorporated dCTP by applying to Sephadex G50.
Hybridization, wash and detection
1) Put the
membrane in a hybridization bottle.
2) Add 5~10
ml of hybridization buffer to 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 immediately 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 in
an 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.
(1)
Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81: 1991-1995.