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E. coli GT116 & GT115
InvivoGen has engineered two specific E. coli strains, named GT116 and GT115,
to facilitate the cloning of shRNAs into psiRNA plasmids. Hairpin structures are
known to be unstable in E. coli due to their elimination by a protein complex
called SbcCD that recognizes and cleaves hairpins. To increase their stability in
E. coli, we developed GT116 and GT115 by deleting the sbcC and sbcD genes.
Both strains are more compatible with hairpin structures than commonly used
E. coli strains increasing the probability of obtaining the correct clone in a
minimum number of steps. GT115 is further mutated in the uidA gene rendering
this strain deficient for b-glucuronidase (see page 42).
Both strains are provided as lyophilized chemically competent cells, named
LyoComp GT116 and LyoComp GT115 respectively, that can also be purchased
separately (see page 43).
GT116 Genotype: F¯ mcrA D(mrr-hsdRMS-mcrBC) Φ80lacZDM15 DlacX74 recA1
rspL (StrA) endA1 ∆dcm ∆sbcC-sbcD
GT115 Genotype: F¯ mcrA D(mrr-hsdRMS-mcrBC) Φ80lacZDM15 DlacX74 recA1
rspL (StrA) endA1 ∆dcm uidA(∆MluI)::pir-116 ∆sbcC-sbcD
Sequencing Primers
Several sequencing primer pairs have been designed, one for each psiRNA
plasmid backbone, that allow full reading of the insert sequence by using
conventional sequencing methods.
- OL559-OL408 pair, for all psiRNA-h7SK plasmids
- OL178-OL408 and OL906-OL176 pairs for the a-peptide cassette and the
GUS cassette of the psiRNA-DUO plasmid, respectively
Fast-Media ® X-Gal & X-Gluc
Fast-Media ® X-Gal and Fast-Media ® X-Gluc are ready-to-use microwaveable
LB-based medium powder for E. coli selection. They already contain the selective
antibiotic and X-Gal (LacZ) or X-Gluc (Gus) at the appropriate co ncentration.
These media allow to prepare high quality white/blue selection medium in less
than 5 minutes.
Fast-Media ® X-Gal is available with four different selections: blasticidin,
hygromycin, kanamycin and Zeocin . Fast-Media ® X-Gluc is available with
Zeocin .
Fast-Media ® can be purchased separately (see pages 44-45).
• Prevalidation of siRNA/shRNA: psiTEST System
The psiTEST System was developed to provide a rapid, simple, and convenient
method to screen for functional siRNA and shRNA sequences (see pages 146-
147). The silencing efficiency of a given siRNA or shRNA is evaluated by
monitoring the reduction of expression of a chimeric gene consisting of a
secreted alkaline phosphatase (SEAP) reporter gene transcriptionally fused to
the target gene. Transfection of effective siRNAs or shRNAs triggers the RNAi
process resulting in the degradation of the chimeric mRNA and therefore the
absence of SEAP activity.
Strategy for psiRNA-mediated RNA
interference
www.invivogen.com/psirna-system 143
RNA INTERFERENCE 7