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pCpGfree-siRNA & pCpGfree-siRNADUO
Description
pCpGfree-siRNA and pCpGfree-siRNADUO are CpG-free plasmids that
allow the expression of one or two shRNAs, respectively. The absence of
CpG dinucleotides in their backbone makes these plasmids less
immunogenic and insensitive to CpG methylation thus ideal for long
lasting expression of shRNA(s) in vivo .
pCpGfree-siRNA
The pCpGfree-siRNA plasmid combines the backbone of the pCpGfree
plasmid with the shRNA expression cassette of the psiRNA plasmid.
pCpGfree-siRNA features the human 7SK promoter, in which all CpG
dinucleotides have been removed. The activity of this RNA Pol III
promoter is augmented by the addition of the enhancer of the murine
CMV immediate-early promoter, a Pol II enhancer 1 . The enhancer has
been modified to remove all CpGs.
The pCpGfree-siRNA plasmid uses Bbs I, an unusual restriction enzyme
that generates asymmetric cohesive overhangs that are not compatible
with each other to eliminate the risk of self-ligation of the vector.
A second cloning strategy can be chosen by using Acc 65I and Hind III
restriction sites.
The pCpGfree-siRNA plasmid exploits the white/blue selection system
to facilitate the screening of recombinant clones. It features an EM7-LacZ
a-peptide cassette between the cloning sites to allow the easy
discrimination between blue parental and white recombinant clones.
pCpGfree-siRNADUO
The pCpGfree-siRNADUO plasmid contains two shRNA cassette driven
by a CpG-free version of the human 7SK promoter. The murine CMV
enhancer has been added to increase the activity of both 7SK promoters.
The first cassette features Acc65 I / Hind III cloning sites and a LacZ apeptide
bacterial expression cassette for white/blue selection in E. coli.
The second cassette contains Bbs I / Bbs I cloning sites and a GUS
bacterial expression cassette, another system that allows white/blue
selection in E. coli.
pCpGfree-siRNA and pCpGfree-siRNA-DUO are provided with
LyoComp GT115, a lyophilized competent E. coli mutant strain permissive
with the white and blue color selection from the LacZ and GUS systems.
Contents and Storage
The pCpGfree-siRNA and pCpGfree-siRNADUO plasmids are provided
as 50 μg of lyophilized DNA in a kit that includes the following
components:
- 20 μg control plasmid
- 1 vial of LyoComp GT115
- 10 μg of each sequencing primer
- 4 pouches of Fast-Media ® Zeo X-Gal or,
- 2 pouches of Fast-Media ® Zeo X-Gal and 2 pouches of Fast-Media ®
Zeo X-Gluc
Products are shipped at room temperature. Store Fast-Media ® pouches
at room temperature. Store all other components at -20°C.
Acc 65I
Bbs I
Important Licensing Information: These products are covered by a Limited Use License (See Terms and Conditions on page 164). By use of these products you
accept the terms and conditions of all applicable Limited Use Label Licenses.
Bbs I
Hind III
Bbs I
PRODUCT QUANTITY CAT. CODE
pCpGfree-siRNA 1 kit kcpgf-sirna
pCpGfree-siRNADUO 1 kit kcpgf-sirna2
Related Products
Acc 65I
LyoComp GT115, page 43 Fast-Media ® Zeo-X-Gal, page 45
Zeocin , page 18 Fast-Media ® Zeo-X-Gluc, page 45
www.invivogen.com/cpgfree-plasmid 137
Bbs I
Hind III
CpG-FREE DNA 6