Biomedical Engineering Reference
In-Depth Information
9.5
S
35
-RNA Probe Synthesis
1. Use 0.5 mL tube or 0.2 mL PCR tube.
2. Add and mix contents below.
1.0
μ
L of purifi ed PCR DNA fragment (0.25
μ
g/
μ
L)
1.0
μ
L of 10× Roche reaction buffer
0.3
μ
L of RNasin [40 U/
μ
L: Promega, cat#N251B]
1.5
μ
L of 10 mM AGC mix solution {
Note *1
}
4.5
μ
L of S
35
-UTP [PerkinElmer, cat#NEG-039H] {
Note *2
}
0.7
μ
L of pure water
1.0
μ
L of T7, T3 or Sp6 RNA polymerase [20 unit/
μ
L: Roche, cat#10881767001,
1031171001, 11487671001, respectively]
3. Incubate the reaction tube immediately at 37 °C for 2 h {
Note *3
}.
4. Fill up to 50
μ
L with 40
μ
L of pure water.
5. Add 2.5
μ
L of 5 M NaCl.
6. Add 125
L of 100 % EtOH, and then mix very well.
7. Incubate the tube at −80 °C or on dry ice at least for 15 min.
8. Centrifuge at max. speed at 4 °C for more than 15 min, and discard
supernatant.
9. Wash pellet with 300
μ
L of 70 % EtOH and centrifuge again at max speed at
4 °C, and discard supernatant.
10. Dissolve pellet with 10
μ
μ
L of pure water by pipetting.
11. Add 40
L of hybridization solution {
Note *4
} and mix well (then store the
solution at −20 °C).
12. For checking radioactive counts, pipette 1
μ
L of S
35
-RNA probe solution into
1 mL scintillation cocktail in a plastic counting vial, and mix very well.
(Add 10
6
cpm of S
35
cRNA probe solution for 100
μ
μ
L hybridization
solution.)
Note
*1: Preparation of 10 mM AGC mix solution.
Mix 2
μ
L of 100 mM ATP [Roche, cat#1140965], 2
μ
L of 100 mM GTP
[Roche, cat#1140957], 2
μ
L of 100 mM CTP [Roche, cat#1140922], and
L of pure water.
Stock mixed solution at −20 °C.
*2: Half-life of S
35
radioactivity is 87.51 days. Therefore, it is better to use
shipped S
35
-UTP solution within 1-1.5 months.
*3: To prohibit condensation formed on top of a tube that affects reaction
effi ciency, use a PCR machine with heat cover.
54
μ
