Bacteriophage M13LR1 and M13LR3 were derivatives of M13mp18 with a 22-base pair (bp) insertion at HindIII site [47 (link)]. M13WX1 and M13X22 were derivatives of M13mp18 with 26 and 22-bp insertion at XbaI site, and phage f1PM-A was a derivative of f1PM with a 27-bp insertion at XbaI site [11 (link)] (Fig. 1 ). E. coli DNA ligase, T4 polynucleotide kinase, HindIII-HFTM and other restriction endonucleases were obtained from New England Biolabs. RecBCD nuclease was purchased from EPICENTRE Biotechnologies. Aphidicolin and lithocolic acid were purchased from Sigma and dissolved in DMSO. Recombinant human MutL-α was kindly provided by Dr. Paul Modrich (Duke University).
E coli dna ligase
Manufactured by New England Biolabs
Sourced in United States
E. coli DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in DNA. It is commonly used in molecular biology workflows involving DNA manipulation, such as cloning and DNA repair.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna polymerase, by New England Biolabs (3 mentions)
Rnase h, by New England Biolabs (3 mentions)
Q5 dna polymerase, by New England Biolabs (3 mentions)
Neb10β, by New England Biolabs (3 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Rnaseh, by Illumina (2 mentions)
Rnase h, by Thermo Fisher Scientific (2 mentions)
Nebuffer, by New England Biolabs (2 mentions)
Dntps, by New England Biolabs (2 mentions)
E coli dna polymerase, by New England Biolabs (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
Aphidicolin, by Merck Group (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
High capacity cdna reverse transcription, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Nextseq 500 mid output v2 kit, by Illumina (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
14 protocols using e coli dna ligase
1
Engineered M13 Phage Derivatives
2
Synthesis of Double-Stranded cDNA from Total RNA
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Total RNA (10 μg) was used to make single-stranded cDNA using High Capacity cDNA Reverse Transcription (Applied Biosystems #4368814) per the manufacturer’s instructions. The second strand was synthesized by adding 30U of E. coli Polymerase I (New England Biolabs #M0209L), 5U of E. coli DNA Ligase (New England Biolabs #M0205S), 5 U of RNase H (Epicentre #R52250), 300 μM of dNTPs (Invitrogen #18427-013) to the first strand reaction. After 2,5 h at 16°C, 5U of T4 DNA polymerase (New England Biolabs #M0203S) was added to the sample and maintaining at 16°C during 40 min. Finally the double-stranded cDNA was cleaned with a QIAquick PCR Purification kit (Qiagen #28104) and quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA). The quantity and quality of all cDNA samples were determined on an Agilent 2100 bioanalyzer.
3
Genomic DNA Removal and cDNA Synthesis
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Genomic DNA (gDNA) was removed from total RNA aliquots extracted without carrier RNA using the Heat & Run gDNA removal kit (ArcticZymes, Tromso, Norway). RNA was reverse transcribed, and first-strand cDNA was synthesized using the NEB Protoscript II first-strand cDNA synthesis kit (New England BioLabs, Ipswich MA) according to the manufacturer’s instructions, followed by second-strand cDNA synthesis using NEB second-strand synthesis enzyme buffer (New England BioLabs, Ipswich, MA) and second-strand DNA enzymes (DNA polymerase I [Escherichia coli ], 10 U; RNase H, 0.35 U; and E. coli DNA ligase, 1.25 U; New England BioLabs, Ipswich, MA). The newly synthesized DNA was purified by ethanol precipitation. DNA libraries were prepared using the Nextera XT DNA library preparation kit (Illumina) and Illumina Nextera XT index kit. The resulting libraries were analyzed, and DNA sizing and quantification were performed using a 2200 TapeStation (Agilent Technologies). A fetal calf serum (FCS) library was prepared as described above from FCS RNA as a negative control. Libraries were diluted to 1 nM, pooled, denatured and diluted to a final concentration of 1.2 pM. Paired-end sequencing was performed using the NextSeq platform (Illumina) using a NextSeq 500 Mid Output V2 kit (Illumina) (21 (link)).
4
Extracting and Purifying Total RNA from Fleas
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Total RNA was extracted from whole fleas or flea tissues in Trizol reagent (Life Technologies, Carlsbad, CA, USA) using a homogenizer (DIAX 600, Heidolph, Schwabach, Germany) and purified following the manufacturer’s protocol. Poly A+ mRNA was enriched from total RNA by oligo-dT cellulose chromatography (Life Technologies). For first strand cDNA synthesis, Poly A+ RNA (5 ug) was reverse transcribed with oligo(dT)20 using Thermoscript (Life Technologies) at 60 °C for 80 min. Second-strand cDNA synthesis was performed in second-strand synthesis buffer (20 mM Tris–HCl [pH 7.4], 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 10 mM DTT, 50 μg/ml BSA), with 150 μM beta-NAD, 5 U of E. coli DNA ligase (New England Biolabs, Ipswich, MA, USA), 3 U of RNase H (Life Technologies), and 40 U of E. coli DNA polymerase I (Life Technologies) in a final volume of 200 ul and incubating the mixture at 15 °C for 2 h and then at 22 °C for 1 h. Double-stranded cDNA was phenol/chloroform extracted, ethanol precipitated in the presence of 4 μg of glycogen carrier, and resuspended in TE buffer (10 mM Tris–HCl [pH 7.4], 1 mM EDTA).
5
Heterologous Expression of mpa BGC
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E. coli strain NEB10β was used for all cloning experiments. E. coli strain WM6026 which was a gift from William Metcalf (University of Illinois at Urbana-Champaign) was used as a conjugation donor for Streptomyces heterologous hosts47 (link). S. albus J1074 and S. lividans TK24 were used for heterologous expression of the cloned mpa BGC. E. coli BL21(DE3) was used for the expression of refactored mpa pathway. M. paraoxydans DSM 15019 was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). S. albus J1074 and S. lividans TK24 were obtained from the Agricultural Research Service Culture Collection (Peoria, IL). E. coli strain NEB10β and BL21(DE3) were purchased from New England Biolabs (Ipswich, MA). Chemicals were purchased from Sigma, Fisher Scientific, or GoldBio. Restriction enzymes, NEBuffers, T4 DNA polymerase, E. coli DNA ligase, dNTPs, NAD+, NEBuilder HiFi DNA assembly mastermix, and Q5 DNA polymerase were purchased from New England Biolabs (Ipswich, MA). All DNA oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA). Codon optimized genes were synthesized by Twist Bioscience (South San Francisco, CA).
6
Nucleosomal DNA Substrate Preparation
7
Second-Strand DNA Synthesis Protocol
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We further added 15 μl 5X second-strand buffer (100 mM Tris-HCl pH 6.9, 450 mM KCl [Sigma-Aldrich #P3911], 23 mM MgCl2 [Sigma-Aldrich # M2670], 0.75 mM β-NAD+ [NEB #B9007S], 50 mM (NH4)2SO4 [Fisher #BP212R-1]), 2 μl 10 mM dA/G/C/UTPs (Promega #U1335), 0.5 μl 1U/μl E. coli DNA ligase (NEB #M0205L), 2 μl 10U/μl DNA Pol I (Enzymatics #P705L), and 0.5 μl 5U/μl RNase H (Enzymatics #Y922L). The mixture was incubated at 16 °C for 2 h and the resulting double-stranded DNA was purified using the MinElute PCR Purification kit with 20 μl EB for elution. After that, the libraries were prepared and sequenced using the same protocol as described in “DNA sequencing”.
8
RNA Purification and cDNA Synthesis
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Briefly, 30 µL of vRNAs or 30 µL of IVT RNA mixes were purified and concentrated to 10 µL using Agencourt RNAClean XP SPRI beads (Beckman Coulter) and subjected to first strand cDNA synthesis using the Superscript III First-Strand Synthesis System for RT-PCR Kit (Thermo Fisher Scientific) and a mixture of random hexamers and U12–U13 specific primers (see above). The second cDNA strand was synthetized using 5 U of E. coli RNase H, 40 U of E. coli DNA polymerase, and 10 U of E. coli DNA ligase for 2 h at 16°C (New England Biolabs). Double-stranded cDNAs were purified using Agencourt RNAClean XP SPRI beads (Beckman Coulter) and quantified using the Quant-iT RNA Assay Kit (Thermo Fisher Scientific).
9
Second Strand DNA Synthesis
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First strand DNA was synthesized using SuperScript III Reverse transcriptase (Invitrogen, Carlsbad, USA) starting with 10 μg of RNA. For second strand DNA synthesis we used 30 units of Escherichia coli DNA Polymerase I (New England Biolabs, Ipswich, USA) in the presence of 2.5 units of RNAse H (Epicentre, Madison, USA), 5 units E. coli DNA Ligase (New England Biolabs, Ipswich, USA), Ligase buffer, DNA Polymerase I Buffer (NEB2) and 300 μM of dNTPs. Water was added to 100 μl and the mixture was incubated for 150 min at 16 °C. Subsequently 5 U of T4 DNA Polymerase was added and incubated for a further 30 min at 16 °C. The final products were cleaned using the cycle pure DNA clean up kit and stored at −20 °C.
10
Heterologous Expression of Natural Products
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E. coli strain NEB10β (New England Biolabs, MA) was used for all cloning experiments. E. coli strains WM6026 and ET12567/pUZ8002 were used as conjugation donors for Streptomyces heterologous hosts. S. avermitilis SUKA17 and S. lividans TK24 were used for heterologous expression of natural product BGCs cloned from Actinomycete strains. B. Subtilis JH642 + sfp was used for heterologous expression of natural product BGCs cloned from Bacillus strains. E. coli conjugation donor WM6026 was a gift from William Metcalf (University of Illinois at Urbana-Champaign). S. avermitilis SUKA17 was a gift from Haruo Ikeda (Kitasato University, Japan). S. griseochromogenes ATCC 14511 was ordered directly from ATCC. All remaining Actinomycete strains were obtained from the Agricultural Research Service (NRRL) culture collection, Peoria, IL. All Bacillus strains were obtained from Bacillus Genetic Stock Center (Columbus, OH). Restriction enzymes, NEBuffers, T4 DNA polymerase, E. coli DNA ligase, dNTPs, NAD+, NEBuilder HiFi DNA assembly mastermix, and Q5 DNA polymerase were purchased from New England Biolabs (Ipswich, MA). All DNA oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA).
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