Biology Reference
In-Depth Information
2. The PCR condition is 35 successive cycles of denaturation at
94 °C for 30 s, annealing at 58 °C for 30 s, and elongation at
72 °C for 2.5 min, followed by a fi nal elongation step at 72 °C
for 10 min.
3. Verify amplifi cation of DNA by agarose gel electrophoresis.
The PCR reaction mixture is directly used as template for in
vitro transcription.
4. Prepare 50
μ
l of transcription mixture as follows: 10
μ
l of 5×
transcription buffer, 6
μ
l of 25 mM NTP mix, 12.5
μ
l of PCR
reaction mixture (template), 0.5
μ
l of RNase inhibitor, 0.625
μ
l
of SP6 RNA polymerase, and 20.375
μ
l of Milli-Q water.
5. Incubate at 37 °C for 3-6 h.
6. Centrifuge the transcription mixture at 20,000 ×
g
for 1 min.
7. Transfer the supernatant to a new centrifuge tube.
8. To the supernatant, add 6.5
μ
l of 7.5 M ammonium acetate
l of ethanol. Mix well and incubate on ice for 15 min.
9. Centrifuge the mixture at 20,000 ×
g
for 20 min at 4 °C.
10. Discard the supernatant, and rinse the pellet with 500
and 125
μ
μ
l of
70 % ethanol.
11. Dry the pellet and dissolve the dried pellet in 22.1
μ
l of Milli-Q
water.
1. Dilute 100 mM retinal with dimethyl sulfoxide to make 10 mM
retinal.
2. Thaw WG extract (WEPRO 7240H), creatine kinase, and dial-
ysis buffer on ice. Keep all reagents on ice while in use. Freeze
promptly afterwards. Prepare 50
3.9 Dialysis-Mode
Synthesis of bR
Proteins by Mixed-
Detergent-
Supplemented WGCF
System
μ
l of translation mixture on
ice as follows: 12.5
μ
l of WG extract, 2
μ
l of 40 mg/ml cre-
atine kinase, 9.4
μ
l of 4× dialysis buffer, 2
μ
l of FC14/CHAPS
mixture, 22.1
μ
l of mRNA, 1
μ
l of RNase inhibitor, and 1
μ
l of
10 mM retinal.
3. Prepare 800
μ
l of substrate mixture on ice as follows: 200
μ
l of
4× dialysis buffer, 32
μ
l of FC14/CHAPS mixture, and 568
μ
l
of Milli-Q water.
4. Transfer the 50
μ
l of translation mixture into a dialysis cup
(
see
Note 9
).
5. Transfer 800
l of the substrate mixture into a receptacle tube.
6. Place the dialysis cup containing the translation mixture into
the receptacle tube.
7. Seal the connected portion with parafi lm to avoid evaporation.
8. Incubate the reaction mixture at 26 °C for 16 h. The typical
yield of dialysis-mode translation is 5-20
μ
μ
g/50
μ
l of transla-
tion mixture.
