Prokaryotic cell in vitro translation system experimental steps - Database & Sql Blog Articles

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―, materials and equipment

1) Nuclease-free water.

2) [35S] labeled methionine (1.25×10-5mol/L12000Ci/mmol)

3) 1 mmol/L amino acid mixture lacking middle methionine

4) S30 reaction mixture lacking amino acids (Promega)

5) S30 extract for cup DNA (Promega)

6) S30 extract for linear DNA (Promega)

7) T7S30 extract for circular DNA (Promega)

8)—70°C refrigerator

9) Micropipette

10) Centrifuge

11) Ice bath

Second, the method of operation

(1) Escherichia coli S30 extract system suitable for in vitro translation of circular DNA

2) Vortex gently and then centrifuge for 5 s to allow the reaction mixture to sink to the bottom of the tube.

3) Incubate for 1 to 2 h at 37 °C.

4) The reaction tube was placed on an ice bath for 5 min to terminate the reaction.

5) Analyze the test results. Three (TCA) immunoprecipitation experiments and SDS-polyacrylamide gel electrophoresis were used to analyze the product,

(2) Escherichia coli S30 extract system suitable for in vitro translation of linear DNA

The specific procedure is the same as 1()) in the E. coli S30 extract system for in vitro translation of circular DNA. The reaction system is shown in Table 4.2.

(iii) E. coli T7S30 extract system suitable for in vitro translation of circular DNA

The specific procedure is the same as "1" in the E. coli S30 extract system for in vitro translation of circular DNA", 5), the reaction system is shown in Table 4.3;