Descriptions of plasmids used in this study are in the Key Resources Table . All sRNAs assayed were overexpressed from pBRplac (Guillier and Gottesman, 2006 (link)). sRNA sequences were PCR amplified using the appropriate primers as listed in Table S9 , digested with AatII and HindIII and cloned into pBRplac digested with the same restriction enzymes. The pBR-RbsZ mutant derivatives were obtained by cloning gBlocks (IDT) with the desired sequences into pBRplac or by site direct mutagenesis using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). ProQ was overexpressed from pBAD33 (Guzman et al., 1995 (link)), a plasmid compatible to pBRplac. The proQ sequence was PCR amplified using primers SM405 and SM406, digested with KpnI and HindIII and cloned into pBAD33 digested with the same restriction enzymes. The sequences of inserts were verified. Construction of rbsB-gfp-fusion plasmids were carried out essentially as described in (Urban and Vogel, 2009 (link)), using the pXG10-SF (Corcoran et al., 2012 (link)) as the backbone. Briefly, the rbsB 5´ region was PCR amplified using primers SM480 and SM481, digested with Mph1103I and NheI and cloned upstream of gfp in pXG10-SF digested with the same enzymes. The pXG10-SF-rbsB mutant derivative was generated by site-direct mutagenesis using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent).
Quikchange lightning site directed mutagenesis kit
Manufactured by Agilent Technologies
Sourced in United States, Germany, Canada, United Kingdom, France, China
The QuikChange Lightning Site-Directed Mutagenesis Kit is a laboratory tool used to perform rapid and efficient site-specific mutations in double-stranded plasmid DNA. The kit provides a reliable and straightforward method for introducing desired sequence changes without the need for subcloning.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (29 mentions)
Dual luciferase reporter assay system, by Promega (17 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (16 mentions)
Gateway system, by Thermo Fisher Scientific (8 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (8 mentions)
In fusion hd cloning kit, by Takara Bio (7 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (7 mentions)
Mmessage mmachine, by Thermo Fisher Scientific (6 mentions)
Quikchange lightning multi site directed mutagenesis kit, by Agilent Technologies (5 mentions)
Bl21 codonplus de3 ripl, by Agilent Technologies (5 mentions)
Dual luciferase reporter assay kit, by Promega (5 mentions)
Psicheck 2 vector, by Promega (5 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Xl10 gold ultracompetent cells, by Agilent Technologies (4 mentions)
Qiaprep spin miniprep kit, by Qiagen (4 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (4 mentions)
Myc ddk tag, by OriGene (4 mentions)
Dual luciferase reporter assay, by Promega (4 mentions)
Pgex 5x 1 backbone, by GE Healthcare (4 mentions)
750 protocols using quikchange lightning site directed mutagenesis kit
1
Plasmid Cloning and Mutagenesis Protocol
2
Cloning and Mutagenesis of GBA1 Gene
3
Evaluating Neutralization Potency Mutations
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To detect changes in neutralisation potency single point mutations were introduced into env encoding plasmids for PV production using the QuikChange Lightning Site-Directed Mutagenesis (SDM) kit (Agilent) according to the manufacturer’s protocol. For neutralisation absorption assays commercially produced MPER peptide (ELLELDKWASLWNWFGITKWLWYIKIFIM, synthesised by Smart BioScience) or in house produced recombinant gp120 D368R was added to serum/mAbs prior to the addition of PV in the neutralisation assay. For competition ELISA, unlabelled mAbs were pre-incubated with blocked lectin immobilised Env. Biotinylated mAbs were then added for 1h, followed by streptavidin-AP detection.
4
Destabilized GFP with miR-1-3p Binding Site
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Destabilized GFP was generated as described by Li et al. [27 (link)]. To insert the 3′UTR counting full complementation miR-1-3p binding site, we used the QuikChange Lightning site-directed mutagenesis (SDM) kit (Agilent, Santa Clara, CA, USA). This kit was also used to create various miRNA canonical sites. The new vectors were verified by Sanger sequencing. Canonical site sequences are listed in Table 1 .
5
Site-Directed Mutagenesis of Transcription Factor Binding Sites
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SDM was performed with QuikChangeTM lightning site-directed mutagenesis kit (Agilent Technologies; Catalog No. 210518), as described in the company’s protocol. Briefly, amino acid exchanges at the Klf9 sites (RKBE 1 and RKBE 3) mutants (RKBE 1; GC to TT and RKBE 3; AC to TT) were produced by point mutations in the human promoter of Prdx6-linked to CAT plasmid as described earlier [11 (link)]. To prepare Nrf2/ARE (ARE 3 and ARE 4) sites mutants, base A was changed to C, and base T to G at ARE sequences in the mouse Klf9 promoter-linked to CAT reporter plasmid by using site-directed mutagenesis. The following complementary primers were utilized (changed nucleotides are in red boldface type and underlined).
6
Site-Directed Mutagenesis of Prdx6 Promoter
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PCR-based site-directed mutagenesis was carried out using the QuikChangeTM lightning Site-Directed Mutagenesis kit (Agilent Technologies; Catalog No. 210518), following the company’s protocol. Briefly, amino acid exchanges at the Bmal1 site (E-Box; -341/-336) mutant (T to A and A to T) and at Nrf2 site (ARE; -357/-349) mutants (TG to GT) were generated by point mutations in the human promoter of Prdx6-CAT plasmid. The following complementary primers were used (changed nucleotides are in red boldface type and underlined):
7
Prdx6 Promoter Characterization via CAT Assay
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The 5′-flanking region (−839 to +109 bp) was isolated from mouse genomic DNA and sequenced. A construct of −839 bp was prepared by ligating it to basic pCAT vector (Promega) using the SacI and XhoI sites. The plasmid was amplified and used for the CAT assay. Primers used were as follows: 5′-CTTCCTCTGGAGCTCAGAATTTAC-3′ and 5′-CAGGAACTCGAGGAAGCGGAT-3′. Sp1 mutants constructs were generated by point mutations in the Prdx6–CAT plasmid using the QuikChangeTM lightning site-directed mutagenesis kit (Agilent Technologies; Catalog No. 210518) as described previously [12 (link)].
8
Site-Directed Mutagenesis of Prdx6 Promoter
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To generate site-directed specific mutation of binding sites of Bmal1/E-Box (5′−341/−336-3′; T changed to A and A changed to T) and Nrf2/ARE (5′−357/−349-3′; TG changed to GT) present in Prdx6 gene promoter (Prdx6-CAT plasmid), we utilized PCR-based site-directed mutagenesis (Catalog No. 210518; QuikChangeTM lightning Site-Directed Mutagenesis kit, Agilent Technologies; Santa Clara, CA, USA) following the company’s protocol and our published method [5 (link),21 (link),81 (link)]. The double-stranded Prdx6 promoter construct (−918/+30) was used as template DNA with a pair of complementary primers to mutate the Prdx6 promoter construct at E-Box and ARE sites. The primers used for mutation were as follows:
9
PCR-based Site-Directed Mutagenesis of Sp1
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PCR-based site-directed mutagenesis was carried out using the QuikChangeTM lightning site-directed mutagenesis kit (Agilent Technologies; Catalog No. 210518), following the company's protocol. Briefly, amino acid exchanges K16R were generated by point mutations in the pCl-neo-HA-Sp1 constructs. The following complementary primers, forward primer: 5′-GCTGTGGTGAGG ATTGAAAAAGGAGTTGGTGGC-3′ and reverse primer: 5’-GCCACCAACTCCTTTTTAAATCC TCACCACAGC-3’ were used (changed nucleotides are in boldface type and underlined).
Epicurean Coli XL1-Blue super-competent cells (Stratagene) were transformed with resultant plasmid. The plasmid was amplified, and the mutation was confirmed by sequencing as described previously [101 (link)].
Epicurean Coli XL1-Blue super-competent cells (Stratagene) were transformed with resultant plasmid. The plasmid was amplified, and the mutation was confirmed by sequencing as described previously [101 (link)].
10
PCR-based Site-Directed Mutagenesis of Nrf2 Site
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PCR-based site-directed mutagenesis was carried out using the QuikChangeTM lightning site-directed mutagenesis kit (Agilent Technologies; Catalog No. 210518), following the company’s protocol. Briefly, amino acid exchanges at the Nrf2 site (ARE; −357/−349) mutant (TG to GT) were generated by point mutations in the human promoter of Prdx6-CAT construct. The following complementary primers were used (changed nucleotides are in boldface type and underlined):
Nrf2-Mutfor 5′-CCAGGGGGCAACGGT
Nrf2-Mutrev 5′-GCACACGTGATGCGGGGCTCGGTAC
Epicurean Coli XL1-Blue super-competent cells (Stratagene) were transformed with resultant plasmid. The plasmid was amplified, and the mutation was confirmed by sequencing as described previously91 (link).
Nrf2-Mutfor 5′-CCAGGGGGCAACG
Nrf2-Mutrev 5′-GCACACGTGATGCGGGGCTCGGT
Epicurean Coli XL1-Blue super-competent cells (Stratagene) were transformed with resultant plasmid. The plasmid was amplified, and the mutation was confirmed by sequencing as described previously91 (link).
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