The plasmid (pET22b(+)) containing the gene of full-length RABV P (CVS-11 strain) (UniProt P22363) fused to a C-terminal two-amino linker and 6xHis-tag for expression in bacteria was previously described [20 (link)]. The cDNA encoding residues from 1 to 68 of RABV phosphoprotein were cloned into the pET28a (Novagen, Darmstadt, Germany) vector using NcoI and XhoI restriction sites in a frame with a downstream 6xHis-tag and a two amino-acid linker (Glu-Leu). A synthetic cDNA (Geneart, Regensburg, Germany) encoding the first 42 residues of RABV phosphoprotein with a cysteine substitution at position 41 (G41C), an N-terminal 6xHis tag, a SUMO tag and a tobacco etch virus protease (TEV) cleavage site were cloned into the pET22b expression vector (Novagen) using the NcoI and XhoI restriction sites. Point mutations were introduced in this construct by site-directed mutagenesis using the QuickChange II protocol (Agilent, Santa Clara, CA, USA). The cDNA encoding residues from 24 to 450 of rabies virus (strain CVS-11) nucleoprotein (UniProt Q8JXF6) were cloned into the pETM-40 (EMBL) vector using NcoI and XhoI restriction sites in frame with the upstream MalE gene encoding the maltose binding protein (MBP) and a TEV cleavage site. All the plasmids were verified using standard dideoxy sequencing.
Quickchange 2 protocol
Manufactured by Agilent Technologies
Sourced in United States
The QuickChange II protocol is a method for site-directed mutagenesis, allowing for the rapid and efficient introduction of mutations into double-stranded plasmid DNA. The protocol enables the creation of point mutations, deletions, and insertions in a wide range of plasmid sizes.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using quickchange 2 protocol
1
Expression and Purification of RABV Proteins
2
Site-directed Mutagenesis of Plasmid DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Site-directed mutagenesis of plasmid DNA was conducted with mutagenic primers and methods based on the QuickChange II protocol (Agilent Technologies, Santa Clara, CA, USA [5 (link)]). The primers, and the mutations they introduce, are listed in Tables S2 and S3 . In some cases, plasmids were constructed using splicing by overlap extension PCR (SOEing) [16 (link)]. Linearized plasmid-borne alleles were used to replace chromosomal genes in A. baylyi recipient strains by homologous recombination [17 (link),18 (link)]. Transformants were identified by phenotypic changes in antibiotic resistance, carbon source utilization or loss of the sacB marker (in the presence of 10% sucrose and no NaCl in the medium) [5 (link),18 (link)]. The genotypes of mutant strains were confirmed by PCR analysis and DNA sequencing (Genewiz laboratories, South Plainfield, NJ, USA) of the chromosomal regions where changes were introduced.
3
Introducing Point Mutations in Nter-HNF1β-GFP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To introduce point mutations in Nter-HNF1β-GFP, we used the QuickChange II protocol (Agilent). In summary, 20 cycles of PCR were carried on pcDNA4TO/Nter-HNF1β with Pfu polymerase (Promega) and a specific set of primers (P256S: 11i122 and 11i123; V265L: 13i94 and 13i95; G287S: 12i64 and 12i65, Supplementary Table S1). Amplicons were ethanol-precipitated and digested over-night by 1 Unit of DpnI at 37°C, and then transformed into DH5alpha cells (Life technologies). Positive clones were sequenced on the full Nter-HNF1β-GFP ORF, and subcloned by a SpeI/BamHI digestion in the original pcDNA4TO/Nter-HNF1β.
4
Site-directed mutagenesis of repABC plasmid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The high copy oriRi plasmid pTF::Ri [21 (link)] was sequenced and assembled by the Monsanto sequence center. This new sequence was aligned with the published pRiA4b sequence (GenBank accession # X04833) using with Needleman-Wunsch algorithm [22 (link)].
The oriRi plasmid pMON83937 [9 (link)], which contains 4114 bp of the repABC sequence from pRiA4b (GenBank X04833, between 7–4120 bp), was used for all site-directed mutagenesis experiments using the QuickChange II protocol (Agilent Cat # 200521). The primers5’ GATCATGTGCCAGCGCTGcAT ATg CAGCGCTGGCACATGATC 3’ (reverse) were used to generate the RepBY299H mutation, whereas the primers 5’ GCAGTTTTCTCGAGAGATc gAT CTCTCGAGAAAACTGC 3’ (reverse) were used to produce the repB T486C silent mutation. Ten ng of pMON83937 was used in a 50 μl reaction volume and amplified for 25 cycles with 5 min elongation time. The reaction mixture was incubated with DpnI to remove the template and transfected into E. coli DH10B competent cells to recover the plasmids. The resultant plasmids were extracted using a Qiagen maxiprep kit and verified by full plasmid sequencing. The plasmids used for this study are summarized in Table 1 .
The oriRi plasmid pMON83937 [9 (link)], which contains 4114 bp of the repABC sequence from pRiA4b (GenBank X04833, between 7–4120 bp), was used for all site-directed mutagenesis experiments using the QuickChange II protocol (Agilent Cat # 200521). The primers
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!