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Cell Free System Protocols

TRANSCRIPTION

SP6 Cell Free Transcription

Set Up Reaction

Order of Setup and Incubation

Reagents

Buffers

Sample Protocol

Transcription Reactions

Troubleshooting

Transcription Kinetics

TRANSLATION

General Cell Free Translation

Reticulocyte Lysate and Wheat Germ Extracts

Rabbit Reticulocyte Lysate Cell-Free Translation

Nuclease Treatment and Desalting

Translation Pausing

Ribosome Pausing Assay

RPFs from Free and Membrane Bound Ribosomes

tRNA Crosslinking

Preparative Aminoacylation of Pure tRNA

Phenol Extraction of tRNA

Generation of Integration Intermediates

Chemical Modification of Lys-tRNA

Immunoprecipitation of translation products

Translation in RRL

Buffers

Sample Protocol

Troubleshooting

Pause Assay

RPFs from Free and Membrane Bound Ribosomes

History and evolution of cell-free protein synthesizing systems

Import Into Mitochondria

Wheat Germ Cell-Free Translation

Protocol for wheat germ cell-free translation

Wheat Germ Extract Buffers and reagents

Sample WG Protocol

Wheat Germ Extract Recipes

Troubleshooting Wheat Germ Translation Reactions

Preparation of a wheat germ extract

E coli Translation

Translation Protocol

Sample Protocol

Troubleshooting

Lysate Preparation of S-30

Membrane-Free Ribosomes (mfrs)

Ribosomal Pellet

Dialysis Tubing Work-up

Membrane-Free Translation System

Buffers and Reagents

TARGETING

Targeting and Translocation

Proteolysis Assays for Translocation

Post Translational Targeting to RMs

Endoglycosidase H

Carbonate Extraction

Separating Membrane-Targeted Molecules using Gel Filtration

Pelleting Assays


Please feel free to try any of our protocols.  They are available in web and Adobe Acrobat format.  All that we ask that you credit us (or the original author) when using them.  This document is in a permanent state of flux - no protocol is ever really perfect and therefore it is never finished!