20 μg of genomic DNA of strain K74 were completely digested with 50 u of the restriction enzyme BamHI (Promega) and electrophoresised. The resulting BamHI fragments with sizes from 1.5 kb to 7.5 kb were recovered from agarose gel using the QIAquick Gel Extraction Kit (Qiagen). The obtained BamHI fragments were ligated into BamHI-cleaved pUC19 vector as follow: 60 ng of purified DNA fragments, 100 ng of pUC19, 2 μl of ligation buffer, and 5 u of T4 ligase (Promega) were mixed; the final reaction volume was adjusted to 20 μl with water. The ligations were performed at 4 °C for 12 h, and the ligation products were desalted using MFTM membrane filters (0.025 μm, VSWP, Millipore) before being electroporated into E. coli strain Trans5α. The transformants were selected on ampicillin-containing LB medium (100 μg/ml) supplemented with IPTG (120 μg/ml) and X-gal (80 μg/ml), the plating density was around 300 colonies per plate (Ø = 14.4 cm). The dot blot on colony was performed following the manual provided with the Amersham Hybond-N+ Membranes (GE Healthcare). The probe used for the hybridization was the same as used in the RFLP analysis. The potential positive clones were confirmed by Southern blot using the same protocol as used in the RFLP analysis, except that the duration of the electrophoresis was 16 h.
Bamhi
Manufactured by Promega
Sourced in United States
BamHI is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 5'-GGATCC-3'. It is commonly used in molecular biology for the purpose of DNA digestion and manipulation.
Automatically generated - may contain errors
Lab products found in correlation
Ecori, by Promega (9 mentions)
Xhoi, by Promega (8 mentions)
Hindiii, by Promega (4 mentions)
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Buffer e, by Promega (3 mentions)
Fragmentation reagent, by Thermo Fisher Scientific (3 mentions)
Circligase 2, by Illumina (3 mentions)
T4 dna ligase, by Promega (3 mentions)
Bglii, by Promega (3 mentions)
Pet28a vector, by Roche (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Bamhi, by Takara Bio (2 mentions)
Ecori, by Thermo Fisher Scientific (2 mentions)
Bamhi, by Qiagen (1 mentions)
Amersham hybond n membrane, by GE Healthcare (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
T4 ligase, by Promega (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Plasmid mini kit, by Qiagen (1 mentions)
43 protocols using bamhi
1
Construction of Genomic DNA Library
2
Construction of pcDNA6/His-mEos2-Rho Vector
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Generation of the pcDNA6/His-mEos2-Rho expression construct was achieved by classical molecular biology techniques. In brief, the coding sequence for mEos2 was extracted from a pSERTa-mEos2 vector (Addgene plasmid 20341) by digestion with BamHI and EcoRI (Promega, Madison, WI) restriction enzymes. The resulting coding DNA fragment was ligated into BamHI and EcoRI digested and alkaline phosphatase (Promega) treated pcDNA6/His-C (Life Technologies, Carlsbad, CA) vector. A c-terminal TGA stop codon carried in by the mEos2 coding sequence was mutated to GGA following a standard Stratagene quick change method. The Rho coding sequence was PCR amplified from a pTAG-RFP-RhoA expression vector (provided by Dr. William Cain) by PFU Ultra II DNA polymerase (Agilent Technologies, Santa Clara, CA) and the following phosphorylated primers: 5’-CCGACCATCCTCCAAAATC-3’ and 5’-GGATCCCTCCAGCAAGGT-3’ (Integrated DNA Technologies, Coralville, IA). The resulting PCR Rho fragment was blunt end ligated into EcoRV digested and alkaline phosphatase treated pcDNA6/his-mEos2 plasmid DNA. The resulting pcDNA6/His-mEos2-Rho vector was confirmed by Sanger sequencing.
3
Extraction and Amplification of Genetic Material
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TRIzol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA), and the one-step RT-PCR kit and Qiagen plasmid mini kit were purchased from Qiagen (Hilden, Germany). BamHI and HindIII were produced by Promega Corp. (Madison, WI, USA), 5-fluorouracil (5-FU) was produced in the Hubei Yuancheng Pharmaceutical Co., Ltd. (Wuhan, China) and recombinant human growth hormone (rhGH) was produced by Merck Serono [(Schweiz) AG (Zug, Switzerland)].
4
Genomic DNA Extraction and Southern Blot Analysis
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Genomic DNA of wild-type and CHAPb Tg mouse tails was extracted in 0.5 ml tail lysisbuffer (50 mM Tris pH 8.0, 100 mM EDTA pH 8.0, 100mM NaCl, 1% SDS and 10 mg/ml prot K) at 55 oC overnight. DNA was precipitated by phenol-chloroform extraction and 10 μg DNA was digested by BamHI (Promega) or XmnI (New England Biolabs) overnight and run on a 1% agarose gel. Probes were generated using the following primers: 5’- AGGGGTCCAGCTCTTTGAAC-3’ and 5’-AGGCTTAAAGCGTCCTCCTC-3’ . The PCR products were then radioactively labelled using α-[p32]dATP (PerkinElmer) by random priming (RadPrime, Invitrogen). DNA blots (Hybond-N+, GE Healthcare) were hybridized with the radioactive probe in ExpressHyb Hybridization buffer (Clontech) and visualized by using a Phosphorimager.
5
Establishment of P. aeruginosa Infection Model
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The P. aeruginosa strain PAO1 was supplied by the Laboratory Department of Tongji Medical Hospital (Huazhong University of Science and Technology, Wuhan, China). Specific pathogen-free (SPF) Ba1b/c mice (n = 60), six to eight-weeks old and weighting 20-30 g, were provided by the Experimental Animal Center in Tongji Medical College. The pGPU6/GFP/Neo- siRNA expression vector for short hairpin RNAs (shRNAs) was purchased from GenePharma (Shanghai, China). The restriction enzymes used for insertion preparation and validation of the shRNA fragments, BbsI, BamHI and PstI, were from Promega (Madison, WI, USA), and the T4 ligase was from Roche (Basel, Switzerland). The plasmid purification kit and reverse transcription (RT) PCR kit were from Promega. The PCR primers were synthesized by Shanghai Invitrogen Bio Co., Ltd. (Shanghai, China). The gel-recovery kit for purification of the final siRNA transfectable products was from TaKaRa (Shiga, Japan). The E-test strip came from AB Biodisk (Solna, Sweden). The antibodies against interleukin (IL-1b and IL-12 cytokines), secondary species-appropriate antibodies, and chemiluminescent etection reagent for use in Western blotting were from R&D Systems (Minneapolis, MN, USA).
6
Extraction and Purification of Environmental DNA
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Recombinant plasmid DNAs pBELX and pNORM1, used as qPCR standards, were extracted using the “Wizard® Plus SV Minipreps DNA Purification System” (Promega) according to the recommendations provided by the manufacturer. The plasmids were linearized by BamHI (Promega) before being purified with the “QIAquick PCR DNA purification kit” (Qiagen). Total environmental DNAs were extracted using the “PowerWater DNA Isolation Kit” (MO BIO laboratories Inc). Briefly, 50 mg of sediments or SMs were thawed before being dispersed in 100 mL of non-pyrogenic sterile water (Aqua B-Braun) by vortexing for 30 s followed by 15 min stirring at 160 rpm and 25°C. These sediment/SM suspensions (or 100 mL of sample for raw waters) were filtered on polycarbonate filters (Whatman Nuclepore filter, pore size 0.22 μm, diameter 47 mm) using a filtration apparatus (Combisart 6-branch Manifold, Sartorius). Total DNAs were directly extracted from the filters according to the recommendations provided by the manufacturer and were eluted from silica columns with 100 μL of PCR grade water (RNase-Free Water, Qiagen). Plasmid and total DNA concentration and purity were estimated by spectrophotometry according to standard procedures, and all DNAs were stored at -20°C until use.
7
RNA-seq library preparation protocol
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10 μg total RNA extracted with Trizol (Ambion) was fragmented with fragmentation reagent (Ambion) at 70°C for 10 min followed by precipitation with ethanol. Reverse transcription was performed with PASSEQ7-2 RT oligo:
and Superscript III (Invitrogen). cDNA was recovered by ethanol precipitation and 120–200 nucleotides of cDNA was gel-purified from 8% urea–PAGE. Recovered cDNA was circularized with Circligase™ II (Epicentre) at 60°C overnight. Buffer E (Promega) was added to the cDNA and heated at 95°C for 2 min, and then cool to 37°C slowly. Circularized cDNA was linearized by BamH I (Promega). cDNA was collected by centrifugation after ethanol precipitation. PCR was carried out with primers PE1.0 and PE2.0 containing index (Illumina). Around 200 bp of PCR products was gel-purified and submitted for sequencing (single read 100 nucleotides).
and Superscript III (Invitrogen). cDNA was recovered by ethanol precipitation and 120–200 nucleotides of cDNA was gel-purified from 8% urea–PAGE. Recovered cDNA was circularized with Circligase™ II (Epicentre) at 60°C overnight. Buffer E (Promega) was added to the cDNA and heated at 95°C for 2 min, and then cool to 37°C slowly. Circularized cDNA was linearized by BamH I (Promega). cDNA was collected by centrifugation after ethanol precipitation. PCR was carried out with primers PE1.0 and PE2.0 containing index (Illumina). Around 200 bp of PCR products was gel-purified and submitted for sequencing (single read 100 nucleotides).
8
Cpn 0810 Gene Amplification and Cloning
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Amplification of Cpn 0810 was performed using polymerase chain reaction (PCR), based on the following primer pairs: P1, 5′-CGCGGATCCATGAATAAAAAGCCCAAGAAAAC-3′, and P2, 5′-TTTTCCTTTTGCGGCCGCTTACTCAGC GCCTTTAACCAT-3′.
Amplification was performed in a final reaction volume of 50 μl, containing 39.6 μl ddH2O, 5 μl 10X Pfu buffer, 1 μl dNTP mix (10mM), 1 μl P1 primer, 1 μl P2 primer, 0.4 μl DNA Polymerase (5 units) and 2 μl Cpn templates. The amplification conditions were as follows: Initial polymerase activation at 94°C (5 min); 30 cycles of 94°C (30 sec), 52°C (45 sec) and 72°C (3 min); and a final elongation step at 72°C for 10 min. Distilled water was used as a negative control. The amplification products (363 bp) were subjected to 1.0% agarose gel electrophoresis containing ethidium bromide.
The PCR products were digested with BamHI and NotI (Promega Corporation, Madison, WI, USA), and ligated into the pGEX6p-2 plasmid (GE Healthcare, Piscataway, NJ, USA). The recombinant plasmid was transformed into E. coli BL21 competent cells, and the positive clones were screened by PCR and sequencing.
Amplification was performed in a final reaction volume of 50 μl, containing 39.6 μl ddH2O, 5 μl 10X Pfu buffer, 1 μl dNTP mix (10mM), 1 μl P1 primer, 1 μl P2 primer, 0.4 μl DNA Polymerase (5 units) and 2 μl Cpn templates. The amplification conditions were as follows: Initial polymerase activation at 94°C (5 min); 30 cycles of 94°C (30 sec), 52°C (45 sec) and 72°C (3 min); and a final elongation step at 72°C for 10 min. Distilled water was used as a negative control. The amplification products (363 bp) were subjected to 1.0% agarose gel electrophoresis containing ethidium bromide.
The PCR products were digested with BamHI and NotI (Promega Corporation, Madison, WI, USA), and ligated into the pGEX6p-2 plasmid (GE Healthcare, Piscataway, NJ, USA). The recombinant plasmid was transformed into E. coli BL21 competent cells, and the positive clones were screened by PCR and sequencing.
9
Arf6 Spatio-Temporal Expression Analysis
10
Optimal Assembly of Yeast Metabolic Pathways
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Expression cassettes and plasmids were assembled in a single reaction using the Gibson Assembly® Master Mix (New England Biolabs—NEB) [85 (link)], and the backbone pRS426 [86 (link)] cleaved with BamHI (Promega). The sequences coding the putative transporters were amplified by PCR using Phusion DNA polymerase (New England BioLabs—NEB) from the gDNA of C. sojae. The control transporter GXF1 was amplified from Candida intermedia genome, using specific primers for each gene (Additional file 10 : Table S6). Sequences of TDH1 promoter and terminator were amplified from S. cerevisiae strain LVA1 genome [10 (link)]. Fragments from PCR amplifications were purified from agarose gel using the Kit Wizard® SV Gel and PCR Clean-Up System (Promega) before the assembly reaction. Cassettes containing the genes XYL1 and XYL2 from S. stipitis and XKS1 from S. cerevisiae were amplified from the plasmids pSsXRXDH and pScXKS (Santos, personal communication). Both cassettes use the URA3 gene flanked by two loxP sites as marker. The correct frame of the gene sequences cloned in the plasmids and expression cassettes were confirmed by Sanger sequencing. The main components of the plasmids are summarized in Additional file 11 : Table S7.
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