Protocols and Applications Guide (US Letter Size) - Promega

||||| 5Protein Expression
Figure 5.9. Structure of FluoroTect GreenLys tRNA.
Use the following protocol as a guideline for setting up
translation reactions using FluoroTect GreenLys tRNA.
In general, FluoroTect GreenLys tRNA may be used in
an in vitro translation protocol at a concentration of 1µl of
FluoroTect GreenLys tRNA per 50µl reaction. Examples
of standard reactions using TNT® T7 Quick for PCR DNA
and Rabbit Reticulocyte Treated Lysate are provided.
1. Assemble the following reactions on ice. Add all
components except the FluoroTect GreenLys tRNA,
and gently mix by pipetting the reaction while stirring
the reaction with the pipette tip. If necessary, spin
briefly in a microcentrifuge to return the sample to the
bottom of the tube. Add the FluoroTect GreenLys
tRNA.
Example Using TNT® T7 Quick for PCR DNA with
FluoroTect GreenLys tRNA
Component
TNT® T7 PCR Quick Master Mix
1mM methionine
PCR-generated DNA template
FluoroTect GreenLys tRNA
Nuclease-Free Water to a final
volume of
Protocols & Applications Guide
www.promega.com
rev. 6/09
Volume
40µl
1µl
2.5–5µl
1–2µl
50µl
Example Using Rabbit Reticulocyte Lysate with
FluoroTect GreenLys tRNA
Component
Rabbit Reticulocyte Lysate,
Nuclease Treated
RNasin® Ribonuclease Inhibitor
Amino Acid Mixture, Complete
FluoroTect GreenLys tRNA
Luciferase Control RNA
Nuclease-Free Water to a final
volume of
Volume
35µl
1µl
1µl
1–2µl
1µl
50µl
2. Incubate at 30°C for 60–90 minutes (conditions will vary
depending on the translation system used).
3. Terminate the reaction by placing on ice. If necessary,
the translation reaction can be stored for several months
at –20°C or –70°C.
Fluorescence Detection
The fluorescent translation product should be resolved by
running a sample on an SDS-PAGE and then visualized by
placing the gel on a laser-based fluorescence scanning
device.
Additional Resources for FluoroTect GreenLys in vitro
Translation Labeling System
Technical Bulletins and Manuals
TB285 FluoroTect GreenLys in vitro Translation
Labeling System Technical Bulletin
(www.promega.com/tbs/tb285/tb285.html)
Promega Publications
PN077 FluoroTect GreenLys in vitro Translation
Labeling System
(www.promega.com
/pnotes/77/9028_23/9028_23.html)
Citations
Shibuya, N. and Nakashima, N. (2008) Characterization of
the 5´ internal ribosome entry site of Plautia stali intestine
virus J. Gen. Virol. 87, 3679–3686 .
The Plautia stali virus contains two open reading frames
and includes a 5´ internal ribosome entry site (IRES) and
an intergenic IRES region. These authors showed that the
5´ IRES was functional and initiated translation in insect
cell lysate but not in rabbit reticulocyte lysate or wheat
germ extract. The efficiency of translation mediated by the
5´ IRES region was tested with and without cap analog
using various firefly and Renilla luciferase reporter
constructs. They also used deletion mutants to identify the
specific regions required for translation initiation.
PubMed Number: 17098985
X. References
Anderson, C. et al. (1983) Preparation of a cell-free
protein-synthesizing system from wheat germ. Meth. Enzymol. 101,
635–44.
PROTOCOLS & APPLICATIONS GUIDE 5-21