E. coli strains were propagated on Lennox agar (LA) or broth (LB) (35 ), and Streptomyces strains were cultivated with LA, LB, and mannitol-soya flour (MS) agar or broth (35 ) Culture medium was supplemented with antibiotics as required at the following concentrations: apramycin, 50 μg/ml; carbenicillin, 100 μg/ml; chloramphenicol, 25 μg/ml; hygromycin, 50 μg/ml; kanamycin, 50 μg/ml; nalidixic acid, 25 μg/ml. Streptomyces strains were constructed by cross-genus conjugation with E. coli as previously described (35 ). Enzymes were purchased from New England BioLabs unless otherwise stated, and oligonucleotides were purchased from Integrated DNA Technologies, Inc. All of the strains, cosmids, and plasmids used in this study are described in Table S1 in the supplemental material, and all of the oligonucleotides and other synthetic DNAs used are provided in Table S2 in the supplemental material.
Oligonucleotide
Manufactured by Integrated DNA Technologies
Sourced in United States, Belgium, India, Singapore
Oligonucleotides are short, synthetic DNA or RNA molecules that serve as building blocks for various applications in molecular biology, genetics, and biotechnology. They are composed of a sequence of nucleotides and can be designed to target specific genetic sequences or to function as probes, primers, or gene expression regulators.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by New England Biolabs (25 mentions)
T4 dna ligase, by New England Biolabs (22 mentions)
γ 32p atp, by PerkinElmer (16 mentions)
Phusion dna polymerase, by New England Biolabs (12 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (12 mentions)
T4 polynucleotide kinase, by New England Biolabs (8 mentions)
Restriction endonuclease, by New England Biolabs (8 mentions)
Ni nta resin, by Qiagen (8 mentions)
Gel extraction kit, by Qiagen (8 mentions)
T4 dna ligase, by Thermo Fisher Scientific (8 mentions)
Bs poly prep columns, by Bio-Rad (7 mentions)
Deoxynucleoside triphosphate, by New England Biolabs (7 mentions)
Quick dna ligase kit, by New England Biolabs (7 mentions)
Sep pak c18 cartridge, by Waters Corporation (7 mentions)
Tryptic soy broth (tsb), by MP Biomedicals (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Dnase 1, by Thermo Fisher Scientific (6 mentions)
Uracil dna glycosylase, by New England Biolabs (6 mentions)
Gibson assembly master mix, by New England Biolabs (5 mentions)
Dntps, by New England Biolabs (5 mentions)
424 protocols using oligonucleotide
1
Cultivation and Genetic Engineering of E. coli and Streptomyces
2
Radioactive Labeling and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following suppliers provided the listed items: [α- and γ-32P]-ATP (3000 Ci/mmol) from Perkin-Elmer (Boston, MA), DE81 filters from Whatman Bio System (Maidstone, England), and Bradford reagents and protein molecular weight markers were purchased from Bio-Rad (Hercules, CA). The oligonucleotides were obtained from the Integrated DNA Technologies (Coralville, IA). An anti-FLAG antibody was obtained from Sigma (St. Louis, MO). The oligonucleotides and the 5’-fluoresent labeled DNA were obtained from the Integrated DNA Technologies (Coralville, IA).
3
Escherichia coli and Streptomyces albus Cultivation
Check if the same lab product or an alternative is used in the 5 most similar protocols
Escherichia coli strains were propagated on Lennox agar (LA) or broth (LB) (42 (link), 43 (link)), and Streptomyces albus S4 strains were cultivated using LA, LB, and mannitol-soya flour (MS) agar or broth (42 (link)). Development of clp mutants was assessed on MS and ISP2 medium (42 (link)). Culture medium was supplemented with antibiotics as required at the following concentrations: apramycin, 50 μg ml−1; carbenicillin, 100 μg ml−1; chloramphenicol, 25 μg ml−1; hygromycin, 50 μg ml−1; kanamycin, 50 μg ml−1; nalidixic acid, 25 μg ml−1. Streptomyces strains were constructed by conjugal mating with E. coli ET12567 as previously described (42 (link)). Enzymes were purchased from New England Biolabs unless otherwise stated, and oligonucleotides were purchased from Integrated DNA Technologies, Inc. All of the strains, cosmids, and plasmids used in this study are described in Table S2 in the supplemental material, and all of the oligonucleotides used are provided in Table S3 .
+ Expand
4
DNA Cloning and Plasmid Construction
5
Recombinant DNA Cloning and Amplification
6
Cloning and Sequencing of ERBB2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oligonucleotides (ordered from Integrated DNA Technologies; listed in S4 Table ) were phosphorylated with T4 Polynucleotide Kinase (NEB M0201S) and annealed. The lentiviral vectors pLKO.1 or pLKO.1_TET (both marked with Hygromycin B) were used to clone the annealed oligos into with T4 DNA Ligase (NEB M0202M). ERBB2 was PCR amplified from pBABEpuro-ERBB2 (Addgene #40978) using T4 PNK treated forward oligo (oACH_001) and a reverse oligo (oTM-795). The resulting PCR product was digested with NotI and inserted into pcDNA3β (gift from Ralph Scully) using EcoRV and NotI cut sites. The plasmids were transformed into Stbl3 E. coli cells (ThermoFischer, C737303) and selected for on LB + Carbenicillin plates. 5mL cultures of single colonies from plates were grown and plasmids extracted with Omega Biotek E.Z.N.A. Plasmid DNA Mini Kit I (D6942-00S). Plasmid sequences were verified via Sanger sequencing or whole plasmid sequencing performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation.
7
Introducing KPC-2 Mutants via QuikChange
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All KPC-2 mutants were created using the QuikChange kit (Stratagene, La Jolla, CA). Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). The following is the list of primers used to introduce mutations (underlined) into pTP123 KPC-2:
P104R: CAAAAATGCGCTGGTTCGC TGGTCACCCATCTC
P104L: CAAAAATGCGCTGGTTCTG TGGTCACCCATCTC
V240A: CGGAACCTGCGGAGCG TATGGCACGGCAAATG
V240G: CGGAACCTGCGGAGGG TATGGCACGGCAAATG
H274Y: CAAGGATGACAAGTAC AGCGAGGCCGTCATC
M49I: CGGTGTGTACGCGATA GATACCGGCTCAG
P104R: CAAAAATGCGCTGGTT
P104L: CAAAAATGCGCTGGTT
V240A: CGGAACCTGCGGA
V240G: CGGAACCTGCGGA
H274Y: CAAGGATGACAAG
M49I: CGGTGTGTACGCG
8
Characterization of B. bacteriovorus Genome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oligonucleotides were from Integrated DNA Technologies (San Diego, California). B. bacteriovorus HD100 genomic DNA was a gift from Dr. John Tudor (Saint Joseph's University). Samples were sequenced at the Yale DNA Analysis Facility on Science Hill (New Haven, CT). Nuclease P1 and amino acids were from Sigma-Aldrich (St. Louis, MO). Phenol, ATP, and chloroform were from Fisher Scientific (Pittsburg, PA). [α-32P]ATP (10 mmol/µCi) was from Perkin Elmer (Shelton, CT). Polyethylenimine (PEI)-cellulose thin layer chromatography (TLC) glass plates were from EMD Millipore (Billerica, MA). Restriction enzymes, Escherichia coli BL21(DE3) and NEB10β strains, OneTaq DNA Polymerase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA). E. coli JF448 was from the Yale Coli Genetic Stock Center (New Haven, CT). E. coli trpA34 was a gift from the Söll Laboratory at Yale University (New Haven, CT).
9
Molecular Biology Reagents and Antibodies Used in Malaria Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The molecular biology reagents were purchased from either MilliporeSigma, USA or Thermo Fisher Scientific, USA, unless otherwise stated. All the restriction enzymes and DNA polymerases were purchased from New England Biolabs, and all the oligonucleotides were purchased from Integrated DNA Technologies, Inc., USA. The following primary antibodies, antisera, and dilutions were utilized: mouse α-tubulin (1:250; Sigma-Aldrich; catalog# T5168), rabbit α-Pfg377 (1:250; kindly gifted by Prof. Pietro Alano at Istituto Superiore di Sanità, Rome, Italy) (29 (link)), and mouse α-PfP230p (1:200, kindly gifted by Prof. Kim C. Williamson, Uniformed Services University of the Health Sciences, USA (28 (link)). Reagents obtained through BEI Resources, NIAID, and NIH include: hybridoma 4B7 α-Pfs25-kilodalton gamete surface protein (Pfs25), MRA-315, contributed by Louis H. Miller and Allan Saul (1:1 in 3% bovine serum albumin/phosphate-buffered saline, mouse). The Alexa Fluor-conjugated secondary antibodies utilized for IFAs were procured from Thermo Fisher Scientific, USA.
10
Yeast Growth Media Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise stated, reagents were purchased from Sigma-Aldrich. Synthetic complete (SC) medium was prepared from 1.4 g L -1 SC drop-out mix lacking uracil, tryptophan, leucine and histidine (Y2001), 6.7 g L -1 yeast nitrogen base without amino acids (Y0626) and 20 g L -1 D-glucose, pH standardized to 5.6. When SC was supplemented with additional amino acids, 60 mg L -1 leucine, 20 mg L -1 uracil, 20 mg L -1 histidine-HCl and 20 mg L -1 tryptophan was added. Yeast Peptone Dextrose medium contained 20 g L -1 D-glucose.
Oligonucleotides were purchased from Integrated DNA Technologies.
Oligonucleotides were purchased from Integrated DNA Technologies.
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!