10 In Situ Hybridization for RNA: Nonradioactive Probe: ss eDNA Probe
145
5. Add equal volume of 24:1 chloroform lisoamyl alcohol and
vortex briefly. Centrifuge 5 mins at 5000 rpm and save upper
aqueous phase.
6. Add 1/10 volume of 3 M sodium acetate, pH 5.5, and 2.5
volume of 100% ethanol. Mix and precipitate overnight at
-20°C or IS mins at -70°C.
7. Centrifuge IS mins at 12000 rpm, and discard supernatant.
8. Dry pellet briefly and resuspend in 1 ml of water.
9. Put the solution in an ultrafiltration unit and centrifuge at
3000 G until 80% is filtrated. Add 800 I.d of water, and centrifuge again. Repeat the centrifugation at least five times in
all. Place still the last remnant at 4°C for more than 1 hr.
10. Measure the optical density of the product by spectrophotometer.
The second PCR amplifies only sense or antisense strand. One Second PCR
PCR tube (100 I·d) generates 2-3).lg of ss eDNA. In order to generate sufficient amount of ss eDNA, prepare some tubes at once
and react them simultaneously.
1. Mix the following in PCR tube on ice:
ds eDNA from first PCR
PCR buffer (Mgz+ free)
50 mM MgClz
10 )JM primer
5 mM dNTP mix
Taq polymerase
DDW
3 pmol ( approximately 1 ng /base)
10 )JI
4.0 )JI
5 )JI
4 )Jl
0.5 )JI (2.5 U)
up to 100 )JI
2. Set the sample tubes on the thermal controller and perform
the following steps:
145
5. Add equal volume of 24:1 chloroform lisoamyl alcohol and
vortex briefly. Centrifuge 5 mins at 5000 rpm and save upper
aqueous phase.
6. Add 1/10 volume of 3 M sodium acetate, pH 5.5, and 2.5
volume of 100% ethanol. Mix and precipitate overnight at
-20°C or IS mins at -70°C.
7. Centrifuge IS mins at 12000 rpm, and discard supernatant.
8. Dry pellet briefly and resuspend in 1 ml of water.
9. Put the solution in an ultrafiltration unit and centrifuge at
3000 G until 80% is filtrated. Add 800 I.d of water, and centrifuge again. Repeat the centrifugation at least five times in
all. Place still the last remnant at 4°C for more than 1 hr.
10. Measure the optical density of the product by spectrophotometer.
The second PCR amplifies only sense or antisense strand. One Second PCR
PCR tube (100 I·d) generates 2-3).lg of ss eDNA. In order to generate sufficient amount of ss eDNA, prepare some tubes at once
and react them simultaneously.
1. Mix the following in PCR tube on ice:
ds eDNA from first PCR
PCR buffer (Mgz+ free)
50 mM MgClz
10 )JM primer
5 mM dNTP mix
Taq polymerase
DDW
3 pmol ( approximately 1 ng /base)
10 )JI
4.0 )JI
5 )JI
4 )Jl
0.5 )JI (2.5 U)
up to 100 )JI
2. Set the sample tubes on the thermal controller and perform
the following steps: