Specifications

CpG-free plasmid backbone

Two RNA polymerase III cloning cassettes

Allows white and blue selection for each cassette

Selectable in E. coli with Zeocin™

Provided with the psiRNA™ tools:
• LyoComp GT115, lyophilized chemically competent E. coli GT115 cells
• Sequencing primers (OL359-OLSI3' and OL499-OL1012 pairs)
• E. coli Fast-Media® Zeo XGal and E. coli Fast-Media® Zeo XGluc

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

pCpGfree-siRNADUO plasmid is provided in a kit that includes the following components:

- 50 μg pCpGfree-siRNADUO plasmid
- 20 μg control pCpGfree-siRNA-LucLac plasmid
- 1 vial of LyoComp GT115 with 2 ml Reconstitutive Solution
- 2 sets of sequencing primers (2 x 10 μg each)
- 2 pouches of E. coli Fast-Media® Zeo X-Gal
- 2 pouches of E. coli Fast-Media® Zeo X-Gluc

Description

CpG-free Plasmid Backbone
All the elements required for replication and selection of the plasmid in E. coli and gene expression in mammalian cells are completely devoid of CpG dinucleotides.

Two RNA Polymerase III Cloning Cassettes
Both RNA Pol III cassettes are driven by a CpG-free version of the human 7SK promoter. To increase the activity of these promoters, the enhancer from the mouse CMV immediate-early promoter has been added .
The first cassette contains the following key elements:
- mCMV enhancer / h7SK promoter
- LacZ α-peptide bacterial expression cassette
- Acc65 I / Hind III cloning sites
The second cassette contains the following key elements:
- h7SK promoter
- GUS bacterial expression cassette
- Bbs I / Bbs I cloning sites
Bbs I is 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.

White/Blue Selection
pCpGfree-siRNADUO exploits the white/blue selection system to facilitate the screening of recombinant clones. It features the LacZ α-peptide and GUS (uidA) reporter genes. GUS encodes β-Glucuronidase an E. coli enzyme capable of hydrolyzing the chromogenic substrate XGluc to generate a blue pigment. Therefore, similarly to LacZ, the expression of GUS in permissive E. coli allows the discrimination between blue parental clones and white recombinant clones.

pCpGfree-siRNADUO is provided with the E. coli GT115, a lacZ uidA mutant strain, enabling the white and blue color selection from the LacZ and GUS systems.

Details