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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN EXPRESSION: In Vitro Translation

IN VITRO TRANSCRIPTION AND TRANSLATION USING THE COUPLED RETICULOCYTE LYSATE SYSTEM

In Vitro Transcription and Translation Using the Coupled Reticulocyte Lysate System
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Overview
This protocol is designed to test random samples on a protein gel. Scale up the reactions accordingly.
 
Procedure
1. Add 0.5 μg of template DNA in 5.5 μl of ddH2O (see Hint #2 and Hint #3).

2. Add 1.0 μl Promega "TNT" reaction buffer.

3. Add 0.5 μl Amino Acid Mixture Minus Methionine

4. Add 0.5 μl RNasin Ribonuclease Inhibitor (40 Units/μl).

5. Add 4.0 μl [35S]-Methionine.

6. Add 1.0 μl DNA Polymerase (T3, T7, or SP6, depending on which promoter is in front of your gene in the template DNA).

7. Add 12.5 μl Rabbit Reticulocyte Lysate.

8. Incubate reaction at 30°C for 90 min.

9. Quick-freeze the reaction and store at -20°C or -80°C.

10. To analyze transcription/translation reactions, add 25 μl of 2X SDS-PAGE Sample Loading Buffer and boil samples for 5 min before loading (see protocol for SDS-PAGE).

Solutions
2X SDS-PAGE Sample Buffer   2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT
125 mM Tris
0.01% (w/v) Bromophenol Blue
20% (v/v) Glycerol
4% (w/v) SDS
RNasin Ribonuclease Inhibitor   40 Units/μl
Amino Acid Mixture Minus Methionine   1 mM each Amino Acid, except Methionine
[35S]-Methionine, 1000 Ci/mmol, 10 μCi/μl   CAUTION! see Hint #1
Rabbit Reticulocyte Lysate
Promega TNT kit
 
BioReagents and Chemicals
RNasin Ribonuclease Inhibitor
Promega TNT kit
Amino Acid Mixture Minus Methionine
Tris
DTT
2-Mercaptoethanol
Bromophenol Blue
Glycerol
SDS
[35S]-Methionine
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Use autoclaved ddH2O or DEPC-treated ddH2O to ensure that the components are nuclease-free.

3. For several reactions, make a "cocktail" mixture to divide over the several microcentrifuge tubes. The individual reactions can then be scaled down to 12.5 μl total volume. Thus, follow the same instructions as in section A above, but add half the amount of components. Incubate reactions for the same amount of time, and analyze by SDS-PAGE.