2013 Promega catalogue
2013 Promega catalogue
2013 Promega catalogue
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Life<br />
Science<br />
Catalog<br />
<strong>2013</strong><br />
Worldwide Contact List<br />
Cell Signaling<br />
psiCHECK-1 and psiCHECK-2 Vectors<br />
Product Size Cat.# Price ($)<br />
psiCHECK-1 Vector 20 μg C8011 464<br />
psiCHECK-2 Vector 20 μg C8021 464<br />
For Research Use Only. Not for Use in Diagnostic Procedures.<br />
Description: The psiCHECK-1 and psiCHECK-2 Vectors are designed<br />
to provide a quantitative and rapid approach for initial optimization of RNA<br />
interference (RNAi). The vectors enable monitoring of changes in expression of<br />
a target gene fused to a reporter gene. In both vectors Renilla luciferase is used<br />
as the primary reporter gene, and the gene of interest is cloned into a multiple<br />
cloning region located downstream of the Renilla translational stop codon.<br />
Initiation of the RNAi process by synthetic siRNAs or in vivo-expressed shRNAs<br />
toward a gene of interest results in cleavage and subsequent degradation of<br />
the fusion mRNA. Measuring decreases in Renilla activity provides a convenient<br />
way of monitoring the RNAi effect. In comparison with other fusion approaches<br />
(e.g., GFP or flag-tags), the Renilla luciferase approach offers more convenient<br />
and rapid quantitation with higher sensitivity. The psiCHECK-1 Vector is<br />
recommended for use in monitoring RNAi effects in live cells. The changes in<br />
Renilla luciferase activity are measured with the EnduRen Live Cell Substrate<br />
(Cat.# E6481), which allows continuous monitoring of intracellular Renilla<br />
luminescence. The psiCHECK-2 Vector contains a second reporter gene,<br />
firefly luciferase, and is designed for endpoint lytic assays. Introduction of firefly<br />
luciferase in the psiCHECK-2 Vector allows normalization of Renilla luciferase<br />
expression, achieving robust and reproducible results.<br />
Features:<br />
• Save Money: Quantitation is performed with a common luminometer;<br />
no need to purchase expensive equipment.<br />
• Choose Your Format: Protocols allow for measurements in live cells or<br />
crude cell lysates.<br />
• Save Time: No requirement for labor-intensive, time-consuming assays<br />
or waiting for phenotypic changes.<br />
• Convenient: No requirement for transfection normalization when using<br />
the psiCHECK-2 Vector.<br />
Storage Conditions: Store at –20°C.<br />
Protocol<br />
siCHECK Vectors<br />
Part#<br />
TB329<br />
BamHI 1738<br />
1674 NotI<br />
1663 PmeI<br />
1654 EcoRI<br />
1651 SmaI<br />
1643 XhoI<br />
1640 SgfI<br />
HSV-TK<br />
promoter<br />
1674 NotI<br />
1663 PmeI<br />
1643 XhoI<br />
1640 SgfI<br />
Synthetic<br />
poly(A)<br />
Amp r<br />
hRluc<br />
BamHI 4451<br />
SV40 Late<br />
poly(A)<br />
hRluc<br />
hluc+<br />
Synthetic<br />
poly(A)<br />
psiCHECK-1<br />
Vector<br />
(3560bp)<br />
hRluc<br />
T7<br />
Promoter<br />
Amp r<br />
psiCHECK-2<br />
Vector<br />
(6273bp)<br />
translation<br />
stop<br />
T7<br />
Promoter<br />
ori<br />
NheI 684<br />
BglII 1<br />
KpnI 58<br />
SV40 early<br />
enhancer/<br />
promoter<br />
HindIII 420<br />
PstI 462<br />
ori<br />
NheI 684<br />
gene of interest<br />
BglII 1<br />
KpnI 58<br />
SV40 early<br />
enhancer/<br />
promoter<br />
4343MA10_3A<br />
4345MA10_3A<br />
5´ 3´ mRNA<br />
+<br />
shRNA<br />
(21–23bp)<br />
5´ 3´<br />
5´ 3´<br />
translation<br />
cleavage<br />
of mRNA<br />
degradation<br />
hRluc<br />
light<br />
hRluc<br />
no light<br />
4339MA10_3A<br />
Section<br />
Contents<br />
Mechanism of action of the psiCHECK Vectors.<br />
Table of<br />
Contents<br />
328<br />
For complete and up-to-date product information visit: www.promega.com/catalog
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