A rapid response system for coupling in vitro transcription with in vitro translation
A rapid response system for coupling in vitro transcription with in vitro translation
TNT 耦联网织红细胞裂解物系统乂可以分为：TNTT7 系统、TNTSP6 系统和 TN 下 PCR 系统。 The TNTT7 system translates proteins from both linear and circular DNA, the SP6 system can only use circular DNA for translation, and the PCR template can be translated using the TNTPCR system.
Operation method
A rapid response system for coupling in vitro transcription with in vitro translation
Materials and Instruments
Plasmid DNA template Move -Materials and equipment Caveat 1) The rate of protein translation by TNT coupled to reticulocyte lysates is template dependent. In a reaction volume of 50ul, 150-500ng of protein is produced from the positive control plasmid.2) The amount of DNA used for a standard reaction is 1ug. 0.2 to 2.0ug of DNA is usually sufficient to obtain satisfactory results. If the amount of DNA is greater than 1ug, it does not increase the protein yield.3) The components of each TNT rabbit reticulocyte lysate system are designed differently. Each component is optimally formulated and substituting a component will decrease the yield. For example, it is not possible to replace SP6RNA polymerase with lower T7RMA polymerase in a given system. Using a different RNA polymerase for a given amount of RNA polymerase in each system will result in a lower yield.4) The insertion fragment of the template for the TNT coupled reticulocyte lysate system must be cloned downstream of the promoter of the SP6, T3, or T7 RNA polymerase, and there is no ATG in the appropriate flipping frame. other sequences that can affect the final yield are sequence effects around the start codon, 5' and 3' untranslated sequences, and 5' cap analogues. Flipping efficiency can be increased by the presence of exciting internal ribosome entry sites such as EMCVmRNA upstream of the coding region or by the presence of Poly(A) at the 3' end of the codon. Addition of the m＇G(5)ppP(5)G analog inhibits translation of TNT-coupled reticulocyte lysates because this free analog binds to translation initiation factors.5) The largest protein that can be obtained by coupling rabbit reticulocyte lysate with TNT coupler is approximately 176kDa.6) In the TNT coupled reticulocyte lysate system, cyclic DNA and all 3 RNA polymerases work well together, whereas linear DNA is only suitable for the T3 and T7 systems, and the translation efficiency with SPSRNA polymerase is greatly reduced. If using linear (e.g. TNTT7 fast coupled transcription/translation system), there should be a 20 bp sequence upstream of the RNA polymerase shoulder motif for effective promoter binding. For more product details, please visit Aladdin Scientific website.
Nuclease-free contaminated water [35S]-labeled methionine TNT Rapid Supermix T7TNTPCR enhancement solution
I 70°C refrigerator Ice bath Micropipettes Centrifuge Water bath Polyacrylamide gel electrophoresis unit
1) Nuclease free contaminated water.
2) [35S] labeled methionine . :1X105mol/L,lOOOCi/mmol
3) Plasmid DNA template
4) TNT Rapid Super Mix: mainly includes TNT-rabbit reticulocyte lysate, TNT reaction buffer, TNTRNA polymerase, nucleoside diphosphate mix, l0 mmol/L arginine and RNasin (30~60U).
5) T7TNTPCR enhancement solution
6) A 70°C refrigerator
7) Ice bath
8) Micro pipette
9) Centrifuge
10) Water bath
11) Polyacrylamide gel electrophoresis unit
II. Methods of operation
(-) In vitro transcription/translation using plasmid DNA as template
1) Remove the TNT Rapid Supermix from a 70℃ refrigerator and hold the reaction tube in your hand so that it melts as quickly as possible. When the TNT Rapid Supermix has melted, place it on ice. The other components can be melted at room temperature and then placed on ice.
2) Referring to the reaction system in Table 4.4, add the reaction mixture to a 0.5 ml or 1.5 ml centrifuge tube and mix by gently blowing up and down with a pipette gun before adding all the components. If necessary, the tube may be centrifuged briefly to bring the reaction mixture to the bottom of the tube.
3) A blank control reaction should be set up without the addition of DNA, which can be used to measure the background of the reaction containing the radiolabeled amino acid.
4) Incubate the reaction tube at 30°C for 60 to 90 min.
5) Analyze the translation results. The results of translation are identified by measuring the radiolabeled amino acids in the product using a scintillation counter and analyzing the translated product by SDS-polyacrylamide gel electrophoresis.
(ii) In vitro transcription/translation using PCR products as templates
Procedure H VIII-) In vitro transcription/translation using plasmids as templates (1) to 5).
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