t7 dna ligase  (New England Biolabs)

Journal: Scientific Reports

Article Title: RyR2/IRBIT regulates insulin gene transcript, insulin content, and secretion in the insulinoma cell line INS-1

doi: 10.1038/s41598-022-11276-8

Figure Lengend Snippet: RyR2/IRBIT regulate methylation of the INS1 and INS2 genes ( a) Schematic of the INS1 and INS2 genes with positions of listed primer pairs. ( b) Number of potential methylation sites (CpG) within the sequence amplified by each primer set. ( c) DNA methylation in listed regions of the INS1 gene in INS-1, IRBIT KO , and RyR2 KO cells were measured by methylation-dependent qPCR (MD-qPCR). The relative fold changes shown are differences in ΔCq values of the target region normalized to the 1-UP1 region, which stays unmethylated between all cell types. ΔCq is the change in the Cq values for FspEI digested DNA relative to Cq values of the respective undigested DNA. **** P

Article Snippet: T4 polynucleotide kinase and T7 DNA ligase were from New England Biolabs (Ipswich, MA).

Techniques: Methylation, Sequencing, Amplification, DNA Methylation Assay, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: RyR2/IRBIT regulates insulin gene transcript, insulin content, and secretion in the insulinoma cell line INS-1

doi: 10.1038/s41598-022-11276-8

Figure Lengend Snippet: Characterization of RyR2 KO cells ( a ) Aligned genomic DNA sequences (exon 6) of the rat RYR2 gene in control and two distinct clones. Sequencing confirmed insertion of an indel (underlined/bolded) leading to a frameshift mutation (bolded) and a premature stop codon (red bolded). ( b ) Preliminary screen (representative of 3 independent experiments) showing caffeine (5 mM) mobilization of Ca 2+ in control INS-1 and an RyR2 KO clone measured with fura-2 AM using a 96-well plate format. Data are shown as mean ± SD. ( c ) Representative experiments showing single-cell imaging of Ca 2+ transients measured using fura-2 AM. RyR2 KO cells are insensitive to stimulation with the RyR2 agonist caffeine (5 mM), whereas caffeine elicits a rapid Ca 2+ transient in INS-1 cells. Both cell lines display a robust Ca 2+ transient in response to the muscarinic agonist carbachol (500 μM). ( d ) Quantitation of the Ca 2+ response (AUC) to 5 mM caffeine in KRBH in control INS-1 and RyR2 KO cells. Lines represent mean ± SD **** P

Article Snippet: T4 polynucleotide kinase and T7 DNA ligase were from New England Biolabs (Ipswich, MA).

Techniques: Clone Assay, Sequencing, Mutagenesis, Imaging, Quantitation Assay

Journal: Nature Communications

Article Title: PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

doi: 10.1038/s41467-022-30355-y

Figure Lengend Snippet: PlasmidMaker overview. Design, Build, Test cycle for construction of plasmids from linear DNA parts. In the first step, a frontend is used to design and choose DNA fragments for assembly. After all the selected sequences passed the quality check, picklists (input for “Build” part) are generated for large-scale synthesis of primers and guides. The received oligos in either 96-well format or 384-well format are transferred into corresponding destination wells for PCR reactions to make specific DNA fragments via liquid handlers. Next, one-pot digestion, ligation, and transformation is performed inside iBioFAB to assemble DNA fragments. Pf Ago-based AREs are used for DNA assembly. In the first step, linear DNA molecules ends are digested with WT and engineered Pf Ago/AREs in a one-pot reaction. The AREs generate 5′ sticky ends of 12 nt length. After purification, the digested DNA molecules are assembled in vitro using a high-fidelity DNA ligase and assembly products are transformed into E. coli cells for screening. Finally, constructed plasmids (input for “Test” part) are checked and the correct plasmids are stocked using the robotic system.

Article Snippet: Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ATP, and Q5 DNA polymerase were purchased from New England Biolabs, MA.

Techniques: Generated, Polymerase Chain Reaction, Ligation, Transformation Assay, Purification, In Vitro, Construct

Journal: Nature Communications

Article Title: PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

doi: 10.1038/s41467-022-30355-y

Figure Lengend Snippet: Analysis of the effects of Pf Ago/AREs recognition sequence GC content and GC-distribution as well as the fragments overall GC content on DNA assembly using Pf Ago/AREs. a 10 randomly generated recognition sequences for both 9 and 12 nt AREs with different GC-contents and GC-distributions were placed on the ends of three sets of linear DNA with different overall GC content to create 60 sets of linear fragments. Each set was then digested by either WT Pf Ago or Pf Ago*/AREs creating 9 or 12 nt sticky ends and assembled by E. coli DNA ligase. v1 and v2 represent different GC-distributions. b Average cleavage/DNA assembly efficiency of both Pf Ago and Pf Ago*/AREs creating 9 or 12 nt sticky ends. Based on these results, Pf Ago/AREs can be programmed to cleave linear DNA ends with a wide range of GC-contents (0-75%). This data was used to help our guide design program to ensure efficient cleavage of DNA ends by Pf Ago/AREs (see Supplementary Text ). Source data are provided as a Source Data file. The assembly efficiency analysis for each set was performed only once.

Article Snippet: Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ATP, and Q5 DNA polymerase were purchased from New England Biolabs, MA.

Techniques: Sequencing, Generated

Journal: Nature Communications

Article Title: PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

doi: 10.1038/s41467-022-30355-y

Figure Lengend Snippet: Overview of the workflow of the automated DNA assembly process. a An online ordering system receives plasmid orders to be assembled. The received plasmid sequence is annotated and processed to generate primers, guides, and liquid handling worklists. b Algorithm for design of DNA guides and choice of enzyme for efficient Pf Ago-based assembly. Using annotated fragments of the plasmid as input, guide search space is created from the junction of the fragments. Based on the GC content of the fragments and guide search space, a suggestion is provided to use either WT Pf Ago and Pf Ago* or only Pf Ago*. The guide library created from the guide search space is scanned to identify 24 bp recognition sequences for high-fidelity plasmid assembly. The recognition sequence is finalized as soon as the guides satisfying the design rules for Pf Ago digestion and Hifi Taq ligation are obtained. c Workflow for generation of liquid handling worklists for Pf Ago-based plasmid assembly. Using reference csv file containing the location of primers/guides in 96-well/384-well plate for each plasmid, liquid handling steps are created for mixing primers and templates, mixing guides, followed by equimolar mixing of purified PCR fragments.

Article Snippet: Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ATP, and Q5 DNA polymerase were purchased from New England Biolabs, MA.

Techniques: Plasmid Preparation, Sequencing, Ligation, Purification, Polymerase Chain Reaction

Journal: Nature Communications

Article Title: PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

doi: 10.1038/s41467-022-30355-y

Figure Lengend Snippet: Characterization of Pf Ago/ARE based DNA assembly capabilities in terms of number of DNA fragments and overall plasmid size. a Analysis of the effects of number of fragments on DNA assembly fidelity and total number of acquired colonies in assembly of the 7.2 kb pAmp-ZeaX plasmid. Assembly fidelity was calculated based on the ratio of yellow colonies to total acquired colonies. All experiments were performed in three biological replicates. b Effects of final product size on assembly fidelity and number of acquired colonies for assembly of plasmid DNA molecules with sizes ranging from 12.3 to 26.8 kb using seven DNA fragments. c Results for assembly of the 26.8 kb plasmid with 8, 9, or 10 DNA fragments. Based on these results, plasmid molecules with sizes up to 27 kb including the ones with sequence repeats can be efficiently assembled by Pf Ago/AREs generating 12 nt sticky ends from up to 10 DNA fragments. All experiments were performed in three biological replicates. For all graphs, error bars for colony number and assembly fidelity, show standard deviation (s.d.) and standard error (s.e.m) respectively. Source data are provided as a Source Data file.

Article Snippet: Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ATP, and Q5 DNA polymerase were purchased from New England Biolabs, MA.

Techniques: Plasmid Preparation, Sequencing, Standard Deviation