The cDNAs for Phrixotrix hirtus red-emitting (PxRE), Pyrearinus termitilluminans green emitting (Pte) and Macrolampis firefly (Mac) luciferases were previously subcloned into pCold-vector (Takara). The cDNA for Phrixotrix vivianii green emitting luciferase (PxGR) was subcloned in pCAN vector. The cDNA of Amydetes vivianii (Amy) and Pyrophorus angustus (Pang) luciferases were cloned in pSport vector (Invitrogen) and the cDNA of Cratomorphus distincus luciferase (Crt) in pBluescript vector (Agilent)35 ,38 (link),57 (link),58 (link),61 (link).
Pbluescript vector
Manufactured by Agilent Technologies
The PBluescript vector is a commonly used plasmid vector for cloning and sequencing applications in molecular biology. It contains a multiple cloning site, an antibiotic resistance gene, and a lac promoter for inducible gene expression.
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4 protocols using pbluescript vector
1
Cloning and Characterization of Diverse Luciferases
2
Cross-species complementation of AtLrgB in P. patens
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For cross-species complementation testing, we used the AtLrgB gene of A. thaliana. AtLrgB cDNA was amplified by RT-PCR from DNaseI-treated total RNA of A. thaliana using the AtLrgB-F0 and AtLrgB-R0 primers, and cloned into the pBluescript vector (Agilent Technologies). The cDNA region was extracted by digestion with EcoRI and BamHI, blunted, and inserted at the EcoRV site between the rice actin promoter and the pea rbcS terminator of the pTKM1 [27 (link)] plasmid. We used the P. patens dynamin-related protein 5B-2 (PpDRP5B-2) genomic region for complementation analysis because disruption of the PpDRP5B-2 gene did not visibly affect P. patens [15 (link)]. The hygromycin phosphotransferase (HPT) gene was inserted into the EcoRV fragment region of the cloned PpDRP5B-2 gene [28 (link)]. AtLrgB cDNA, with the promoter and terminator, was isolated by digestion with XbaI and KpnI, subjected to blunting, and inserted into the NheI site of the cloned PpDRP5B-2 gene bearing the HPT gene; P. patens transformation followed (S5 Fig .). PEG-mediated transformation was performed using PpLrgB1 knockout line #2. Insertion of AtLrgB cDNA in the PpDRP5B-2 region, and expression thereof, were confirmed by Southern and RT-PCR analyses (S5 Fig .).
3
Whole-mount X-gal staining and in situ hybridization of mouse embryos
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Tetraploid blastocysts were produced by electrofusion, and ES cells injected to make entirely ES-cell-derived embryos by tetraploid complementation (Nagy et al., 1993 (link)). Whole-mount X-gal staining of embryos was performed at E11.5 as described previously (Lettice et al., 2003 (link)), but on this occasion the staining was allowed to proceed at room temperature for between 1 and 18 h in a concentration of 300 µg/ml X-gal.
Wild-type mouse embryos were harvested at E11.5 and in situ hybridisation was performed with DIG-labelled gene-specific antisense probes as previously described (Hecksher-Sorensen et al., 1998 (link)).
Probes were generated for Lmbr1, Rnf32, Nom1 and Rbm33 by RT-PCR and cloned into the pBluescript vector (Agilent Technologies) (primers are listed insupplementary material Table S1 ). The Shh probe was kindly provided by Andy McMahon (Echelard et al., 1993 (link)) and the Cnpy1 and En2 probes by Jean M Herbert (Paek et al., 2012 (link)).
Wild-type mouse embryos were harvested at E11.5 and in situ hybridisation was performed with DIG-labelled gene-specific antisense probes as previously described (Hecksher-Sorensen et al., 1998 (link)).
Probes were generated for Lmbr1, Rnf32, Nom1 and Rbm33 by RT-PCR and cloned into the pBluescript vector (Agilent Technologies) (primers are listed in
4
Cloning and Transcription of Hsp70 mRNA Variants
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A series of T7 RNA transcription constructs containing variations in the 5′ end sequence of mouse Hsp70 mRNA were cloned into the pBluescript vector (Agilent) using Q5 Site-Directed Mutagenesis Kit (NEB). The resulting plasmids were then overexpressed, cleaved to the appropriate length with restriction enzyme BamHI (NEB) and then used for in vitro transcription of RNA constructs used in this study. Specifically, plasmids pUTR1 [1–231, numbering is the span from the mRNA 5′ end (position 1) to the position within the gene (position 231 in this case)], pUTR2 (1–261), pUTR4 (1–285), pUTR7 (1–331), pUTR9 (1–381), pUTR10 (1–315), pUTR21 (1–372), pUTR22 (1–363), pUTR23 (1–354), pUTR24 (1–345), pUTR25 (1–336) and pUTR26 (1–327) encode the corresponding RNAs used in this study. The unzipping and compensatory RNA mutants are encoded by plasmids pM9a-2 (H1 unzipping mutant 1a) and pM9a-2C (H1 compensatory mutant 1b), pM9e (H1 unzipping mutant 2a) and pM9eC (H1 compensatory mutant 2b), pM7f (H4 unzipping mutant 1a) and pM7fC (H4 compensatory mutant 1b), pM7h (H4 unzipping mutant 2a) and pM7hC (H4 compensatory mutant 2b), pM1-2 (H6 unzipping mutant 1a) and pM1-2C-AUG (H6 compensatory mutant 1b), and pM5-2 (H6 unzipping mutant 2a) and pM5-2C-AUG (H6 compensatory mutant 2b).
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