Protocols and Applications Guide (US Letter Size) - Promega
Protocols and Applications Guide (US Letter Size) - Promega
Protocols and Applications Guide (US Letter Size) - Promega
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||||| 5Protein Expression<br />
St<strong>and</strong>ard Reaction Conditions Using [35S] Methionine, Transcend tRNA or FluoroTect GreenLys tRNA.<br />
Components<br />
TNT® Quick Master Mix<br />
Methionine, 1mM (mix gently prior<br />
to use)<br />
[35S]methionine 1,000Ci/mmol at<br />
10mCi/ml)<br />
plasmid DNA template (0.5µg/µl)<br />
Transcend Biotin-Lysyl-tRNA<br />
FluoroTect GreenLys tRNA<br />
Nuclease-Free Water to a final<br />
volume of<br />
PCR-Generated DNA Templates<br />
For PCR-generated templates, the T7 TNT® PCR Enhancer<br />
should be included in the transcription/translation reaction.<br />
Assemble the reaction components (below) in a 0.5ml or<br />
1.5ml microcentrifuge tube. After addition of all the<br />
components, gently mix by pipetting. If necessary,<br />
[35S]<br />
methionine<br />
40µl<br />
–<br />
2µl<br />
2µl<br />
–<br />
–<br />
50µl<br />
Transcend<br />
tRNA<br />
40µl<br />
1µl<br />
–<br />
2µl<br />
1–2µl<br />
–<br />
50µl<br />
FluoroTect<br />
GreenLys<br />
tRNA<br />
40µl<br />
1µl<br />
–<br />
2µl<br />
–<br />
1–2µl<br />
50µl<br />
centrifuge briefly to return the reaction to the bottom of<br />
the tube. For the control reaction, use 1µl of the Luciferase<br />
Control DNA supplied.<br />
Note: We recommend also including a negative control<br />
reaction containing no added template to allow<br />
measurement of background incorporation of labeled amino<br />
acids.<br />
St<strong>and</strong>ard Reaction Conditions Using PCR-Generated Templates with [35S]Methionine, Transcend tRNA or<br />
FluoroTect GreenLys tRNA.<br />
Components<br />
TNT® Quick Master Mix<br />
Methionine, 1mM (mix gently prior<br />
to use)<br />
[35S]methionine 1,000Ci/mmol at<br />
10mCi/ml)<br />
PCR-generated DNA template<br />
T7 TNT® PCR Enhancer<br />
Transcend Biotin-Lysyl-tRNA<br />
FluoroTect GreenLys tRNA<br />
Nuclease-Free Water to a final<br />
volume of<br />
Additional Resources for Eukaryotic Cell-Free Expression<br />
Systems<br />
Technical Bulletins <strong>and</strong> Manuals<br />
TM282 TNT® SP6 High-Yield Protein Expression<br />
System Technical Manual<br />
(www.promega.com<br />
/tbs/tm282/tm282.html)<br />
TM305 TNT® T7 Insect Cell Extract Protein<br />
Expression System Technical Manual<br />
(www.promega.com<br />
/tbs/tm305/tm305.html)<br />
<strong>Protocols</strong> & <strong>Applications</strong> <strong>Guide</strong><br />
www.promega.com<br />
rev. 6/09<br />
[35S]<br />
methionine<br />
40µl<br />
–<br />
2µl<br />
2.5–5µl<br />
1µl<br />
–<br />
–<br />
50µl<br />
Transcend<br />
tRNA<br />
40µl<br />
1µl<br />
–<br />
2.5–5µl<br />
1µl<br />
1–2µl<br />
–<br />
50µl<br />
FluoroTect<br />
GreenLys<br />
tRNA<br />
40µl<br />
1µl<br />
–<br />
2.5–5µl<br />
1µl<br />
–<br />
1–2µl<br />
50µl<br />
TB165 TNT® Coupled Wheat Germ Extract Systems<br />
Technical Bulletin<br />
(www.promega.com/tbs/tb165/tb165.html)<br />
TM045 TNT® Quick Coupled<br />
Transcription/Translation Systems Technical<br />
Manual<br />
(www.promega.com<br />
/tbs/tm045/tm045.html)<br />
<strong>Promega</strong> Publications<br />
PN058 The TNT® T7 Quick Coupled<br />
Transcription/Translation System<br />
(www.promega.com<br />
/pnotes/58/5189b/5189b.html)<br />
PROTOCOLS & APPLICATIONS GUIDE 5-7
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