psiRNA Tools
psiRNA-h7SK plasmids are provided in a kit that contains, in addition to the cloning vector, two control vectors, and a set of tools designed to facilitate the cloning of shRNAs in the plasmid.
The psiRNA-h7SK tools are the following:
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 β-glucuronidase.
Both strains are provided as lyophilized chemically competent cells, named LyoComp GT116 and LyoComp GT115 respectively, that can also be purchased separately.
GT116 Genotype: F¯ mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 Δdcm ΔsbcC-sbcD
GT115 Genotype: F¯ mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 Δdcm uidA(ΔMluI)::pir-116 ΔsbcC-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 α-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 concentration. 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.