In vitro translation system for prokaryotic cells
In vitro translation system for prokaryotic cells
The S30 family of extracts is divided into three categories: the E. coli S30 Extract System for in vitro translation of circular DNA, the E. coli S30 Extract System for in vitro translation of linear DNA, and the E. coli T7S30 Extract System for in vitro translation of circular DNA.
Operation method
In vitro translation system for prokaryotic cells
Materials and Instruments
S30 extract for cup DNA S30 extract for linear DNA T7S30 extract for circular DNA Move -Materials and equipment Caveat 1) Optimize the amount of DNA. Normally, a reaction system should not contain more than 4ug of DNA, because the increasing amount of DNA will lead to the production of more target proteins without the labeled amino acid, the increase of translation reactions from within the sequence, and the increase of over-translated products.2) Since S-labeled amino acids are easily bismuthated to sulfoxide and become inactive, [35S]-labeled methionine should be stored in portions at 70°C and used after each melting to obtain the best reaction results.3) The purity of template DNA and water is very important. If translation efficiency is low, the purity of the water and template should be checked.4) The reaction can be carried out in the temperature range of 24 to 37°C, with the fastest reaction at 37°C, which takes about 2 h. Lower temperatures result in a lower catalytic rate and a corresponding increase in reaction time by several hours. If lh at 37°C does not yield the expected results, a longer reaction time at lower temperatures should be attempted to increase the amount and specificity of protein expression. For more product details, please visit Aladdin Scientific website.
Nuclease-free water [35S]-labeled methionine 1 mmol L Amino acid mixture lacking mesothionine S30 reaction mixture lacking amino acids
-70°C Refrigerator Micropipettes Centrifuge Ice baths
1) Nuclease free water.
2) [35S] labeled methionine (1.25×10-5mol/L12000Ci/mmol)
3) 1 mmol/L mixture of amino acids lacking mesothionine
4) S30 reaction mixture lacking amino acids (Promega Corporation)
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) Micro pipette
10) Centrifuge
11) Ice bath
II. Methods of operation
(i) E. coli S30 Extract System for In Vitro Translation of Cyclic DNA
2) Gently vortex and centrifuge for 5s to allow the reaction mixture to settle to the bottom of the tube.
3) Incubate at 37℃ for 1 to 2 h.
4) Place the reaction tube on an ice bath for 5 min to terminate the reaction.
5) Analyze the results. The products were analyzed using trichloroacetic acid (TCA) immunoprecipitation assay and SDS-polyacrylamide gel electrophoresis.
(ii) E. coli S30 Extract System for In Vitro Translation of Linear DNA
The procedure is the same as 1) to 5) in "(I) E. coli S30 Extract System for In Vitro Translation of Cyclic DNA". The reaction system is shown in Table 4.2.
(iii) E. coli T7S30 Extract System for the in vitro translation of cyclic DNA 迸.
The procedure is the same as 1) to 5) in "I) E. coli S30 Extract System for In Vitro Translation of Cyclic DNA", and the reaction system is shown in Table 4.3;