sali hf  (New England Biolabs)

Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

Article Title: Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

doi: 10.1002/0471142727.mb1216s103

Figure Lengend Snippet: Restriction map of a FLASH plasmid harboring an extension unit Extension units are released from their plasmid vectors using a quadruple digest. In the example shown, the extension unit contains coding sequence for four TALE repeats (colored rectangles). Digestion of the plasmid with BbsI and BamHI releases the unit from the plasmid with appropriate overhangs for use in the FLASH assembly method. Digestion of the plasmid with the additional restriction enzymes XbaI and SalI prevents the released unit fragment religating back into the vector, thereby eliminating the need to gel purify the fragment.

Article Snippet: ) 100X Bovine Serum Albumin (BSA) (10 mg/ml) (NEB cat. no. B9001S) Restriction enzymes (NEB): BamHI-HF (cat. no. R3136L) XbaI (cat. no. R0145L) BbsI (cat. no. R0539L) SalI-HF (cat. no. R3138L).

Techniques: Plasmid Preparation, Sequencing

Journal: PLoS Biology

Article Title: The dinucleotide composition of the Zika virus genome is shaped by conflicting evolutionary pressures in mammalian hosts and mosquito vectors

doi: 10.1371/journal.pbio.3001201

Figure Lengend Snippet: Fitness comparison between WT and mutant ZIKV in human and mosquito cells. Human A549 cells, A549 ZAP knockout cells, and AP-61 mosquito cells were infected with equal RNA concentrations of WT ZIKV mixed with either the SCR (A) , CpG-high viruses (CpG_1.0 and CpG_max) (B, C), or UpA_max (D) . Virus was passaged to new cells twice, before total RNA was isolated. ZIKV RNA was amplified with RT-PCR and digested with BsaI (A), SalI (B, C), or HeaIII (D) restriction enzymes to distinguish between WT and the respective mutant viruses. DNA gel images display the largest most distinctive products from each digestion. For more information, see S3 Fig . From left to right images display the digested DNA of the indicated individual viruses, followed by 2 independent experimental repeats performed with separately rescued virus populations (numbered 1 and 2). RT-PCR, reverse transcription PCR; SCR, scrambled control virus; WT, wild-type; ZAP, zinc-finger antiviral protein; ZIKV, Zika virus.

Article Snippet: The resulting DNA amplicons from the comparisons between WT and SRC were digested with BsaI-HFv2 (New England Biolabs), WT and CpG_1.0 or CpG_max with SalI-HF (New England Biolabs) and WT and UpA_max with HaeIII (Invitrogen) and ran on 2% agarose gels.

Techniques: Mutagenesis, Knock-Out, Infection, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

doi: 10.1101/2020.05.17.099622

Figure Lengend Snippet: EFF-1 expression in BWM enables VSVΔG-AFF-1 and VSVΔG-G spreading along fused muscles (A-L) Z-stack projections of wt-like (A-F) and Unc+Dpy animals (G-L) expressing myo-3p:: EFF-1 and myo-3p:: mCherry infected with VSVΔG-AFF-1 (35-63 IU red pins) or VSVΔG-G (3*10 5 IU; white pins). Insets and their corresponding images (yellow frames). Arrows, individual infected (cyan) BWMs. Dashed lines outline grouped BWMs that express myo-3p ::mCherry and myo-3p:: EFF-1 (magenta) and infected with virus (cyan) showing spreading of GFP. Scale bars, 100 µm. (M-N) Number of BWM cells/worm expressing EFF-1 (magenta cell), infected (cyan cell) or expressing EFF-1 and infected (magenta and cyan). wt-like (circles) and Unc+Dpy (triangles). Each point represents a single worm. (O-P) Quantitation of multinucleation of infected BWMs. Each dot represents an average number of nuclei/ GFP(+) BWM, calculated from 1-6 multinucleated BWMs of a single worm. (M and O) wt-like n=6 and Unc+Dpy n=10 animals. (N and P) wt-like n=9 and Unc+Dpy n=7 animals. Black horizontal lines, average ± SEM. Student’s t-test, *** p

Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

Techniques: Expressing, Infection, Quantitation Assay

Journal: bioRxiv

Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

doi: 10.1101/2020.05.17.099622

Figure Lengend Snippet: EFF-1 expression in BWMs produces Unc+Dpy worms with multinucleated cells (A-H) Images of fluorescent Z-stack projections and respective DIC of animals with extrachromosomal myo-3p ::EFF-1, myo-3p ::mCherry. (G) White arrowhead, bridge formed between 2 BWMs from opposing quadrants. Yellow arrows, myo-3p ::mCherry accumulations also in DIC (H). Asterisks, clustered nuclei within one BWM. Scale bars, 100 µm. (I) Number of nuclei per myo-3p ::mCherry (+) BWM cell in L2s. wt-like (n=12); Unc+Dpy (n=15). Each dot represents the average number of nuclei/BWM cell/worm. Total average ± SEM for each phenotype. Two tailed Student’s t-test p

Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

doi: 10.1101/2020.05.17.099622

Figure Lengend Snippet: VSVΔG-AFF-1 does not infect PVD and other sensory neurons ectopically expressing AFF-1/EFF-1 (A-C) SDC microscope Z-stack projections of animals infected with 82-103 IU VSVΔG-AFF-1 (red pins). (For genotypes and quantitation see Table S2). Scale bar, 100 µm. (A) Young adult expressing mCherry in PVD. (B) eff-1(ts) adult expressing AFF-1 in PVD. (C) eff-1(ts) adult expressing EFF-1 and dsRed in 12 sensory neurons. See also Table S2 and Movie S2.

Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

Techniques: Expressing, Microscopy, Infection, Quantitation Assay

Journal: bioRxiv

Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

doi: 10.1101/2020.05.17.099622

Figure Lengend Snippet: EFF-1 expression in BWMs induces their fusion ( A-C ) Confocal images of wt-like adult worms with membrane bound ( MB ) myo-3p::MB::YFP (cyan) and extrachromosomal array containing myo-3p:: EFF-1, myo-3p:: mCherry (magenta). ( D-F ) Confocal images of Unc+Dpy [ myo-3p::MB::YFP (cyan); myo-3p::EFF-1, myo-3p::mCherry] . Arrows, unfused BWMs with MB (cyan). Note only two unfused BWMs, all the others appear fused with no MB separating them. Insets correspond to white-dotted area. Scale bars 100 µm. See also Movie S1.

Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

Techniques: Expressing

Journal: bioRxiv

Article Title: EFF-1 promotes muscle fusion, paralysis and retargets infection by AFF-1-coated viruses in C. elegans

doi: 10.1101/2020.05.17.099622

Figure Lengend Snippet: Retargeting of VSVΔG-AFF-1 to body wall muscle cells Wild-type worms and animals with extrachromosomal array containing myo-3p:: EFF-1 and myo-3p:: mCherry were injected with VSVΔG-AFF-1 (35-63 IU, red pins; n=39 wt, n=50 wt-like and n=27 Unc+Dpy worms) or VSVΔG-G (3*10 5 IU, white pins; n=30 wt-like and 14 Unc+Dpy) respectively. Wt worms injected with VSVΔG-G (2300-4700 IU, n=56) were taken from figure 2I. Animals were analysed by SDC microscopy. Data represents average percentage of worms with GFP(+) BWMs ± SEM. Student’s t-test: *p

Article Snippet: DNA constructs The myo-3p ::EFF-1 plasmid was constructed by cloning the myo-3 promoter region from myo-3p ::mCherry plasmid with Sal I (New England BioLabs Cat#R3138) and Nhe I (ThermoFisher Cat# FD0974) and inserting it into the hsp16-2 :: EFF-1 plasmid cut with the same enzymes to replace the original heat shock promoter.

Techniques: Injection, Microscopy

Journal: bioRxiv

Article Title: gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae

doi: 10.1101/2021.02.11.430729

Figure Lengend Snippet: Molecular cloning of chiA-3 and expression of ChiA-3-6xHis. (a): Schematic of cloning strategy used to amplify and insert chiA-3 directionally into the pBAD33 multiple cloning site (MCS), under the arabinose-inducible P BAD promoter, and to incorporate a C-terminal 6xHis tag as a translational fusion. A linker sequence was not incorporated between the C-terminus of ChiA-3 and the 6xHis tag. Figures are not to scale. (b): InstantBlue-stained acrylamide gel of proteins present in supernatants and cell pellet lysates from cultures grown at 23 °C supplemented with arabinose (induction +) or glucose (induction -). No induced bands were easily discerned. (c): Western immunoblot produced from an identically-loaded acrylamide gel to that presented in (b), run in parallel with the gel in (b), and probed with an α-6xHis antibody (see Methods). A band corresponding to the expected molecular weight of ChiA-3-6xHis (48.51 kDa) was detected in the cell pellet lysate of E. coli harbouring pMJD157 only (plasmid +). This size is consistent with the retention of the fusion protein without the cleavage of the putative signal sequence. Protein ladders: NEB #P7719S and #P7717S. EV = empty vector (pBAD33).

Article Snippet: The amplicon was purified and digested using 30 units of SacI-HF and SalI-HF (NEB, #R3156S and R3138S respectively) at 37 °C for 45 min. pBAD33 was similarly treated with SacI-HF and SalI-HF, and after 15 min incubation at 37 °C, the plasmid digestion was supplemented with 1.5 units of recombinant shrimp alkaline phosphatase (rSAP; NEB #M0371S).

Techniques: Molecular Cloning, Expressing, Clone Assay, Sequencing, Staining, Acrylamide Gel Assay, Western Blot, Produced, Molecular Weight, Plasmid Preparation