DNA of the donor strains, plasmid curing mutants and transconjugants was isolated with the NucleoSpin plasmid DNA kit (Macherey-Nagel) and 5 ng was used for PCR amplification with OneTaq DNA polymerase (New England Biolabs). The presence of the three Phaeobacter inhibens D. shibae
Nucleospin plasmid dna kit
Manufactured by Macherey-Nagel
Sourced in France
The NucleoSpin plasmid DNA kit is a laboratory product designed for the purification of plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to efficiently bind, wash, and elute plasmid DNA from cell lysates.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (2 mentions)
Onetaq dna polymerase, by New England Biolabs (1 mentions)
Nucleospin microbial dna kit, by Macherey-Nagel (1 mentions)
Nextera dna flex library prep kit, by Illumina (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Basespace, by Illumina (1 mentions)
4150 tapestation system, by Agilent Technologies (1 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
Pet30a vector, by Merck Group (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Mastercycler pro, by Eppendorf (1 mentions)
6 protocols using nucleospin plasmid dna kit
1
Plasmid Isolation and Validation
2
Plasmid DNA Isolation and PCR Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmid DNA of the transconjugants was isolated with the NucleoSpin Plasmid DNA kit (Macherey-Nagel) and used for PCR amplification with the Crimpson Taq DNA polymerase. Two specific primer sets were established for the 191-kb as well as the 126-kb plasmid that allow the differentiation between the two sister plasmids: PCR-A pDSHI01 (191-kb), P450 (5′-TTACGAAAAACCCGCAGAAGG-3′), and P451 (5′-CCGTTGACCCTTTCGTGGCTG-3′); PCR-B pDSHI01 (191-kb), P430 (5′-TCTGGCTGCGTGGTGGCTTTC-3′), and P431 (5′-TGCGCTATAGTGCTCTCAACA-3′); PCR-C pDSHI03 (126-kb), P432 (5′-GGCACCATCGTCGGAACCAAT-3′), and P433 (5′-TGGTATCAGGCATTCGCTTCA-3′); PCR-D pDSHI03 (126-kb), P059 (5′-CTGACCGTGTTGGAAAGAAGT-3′), and P064 (5′-GCACGAAAAGGCAAAAGA-3′). PCR amplification of the 262-kb plasmid of P. inhibens with P100 (5′-AAACCTTCGTGCCGCTTGTGA-3′), and P105 (5′-CCCAGTTGGAGGATGAGG-3′) served as a positive control (PCR-E). PCR reactions A, B, C, and E were performed with 1 ng of purified plasmid DNA, 5 ng were used for reaction D.
3
NGS Library Prep and Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA from KP5825 was isolated by NucleoSpin Microbial DNA Kit (Macherey Nagel), and plasmid DNA was isolated by NucleoSpin Plasmid DNA Kit (Macherey Nagel) according to the manufacturer’s instructions. The quality and quantity of isolated DNA was assessed by measurements using a Qubit 4.0 fluorometer (Invitrogen, Waltham, USA) and Tapestation 4150 systems (Agilent, Santa Clara, USA). The NGS libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina, Eindhoven, The Netherlands) with Nextera DNA CD Indexes45 (link). The NGS libraries were sequenced on an Illumina MiSeq instrument using the MiSeq Reagent Kit v2 using paired end 250 bp reads at the Genomics Resource Center at the Biomi Ltd. The fastq files were imported directly from Illumina BaseSpace to the BioNumerics version 7.6 software’s (Applied Maths NV, Belgium) cloud-based calculation engine45 (link). De novo sequence assemblies were made with the SPAdes de novo genome assembler (version 3.7.1).
4
Cloning of the agltp24 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The agltp24 gene was PCR amplified from cDNA prepared above using designed primers (Table S1) under the following conditions: 98 °C for 30 s; 30 cycles of 98 °C for 7 s, 63 °C for 20 s and 72 °C for 8 s. The reaction mixture of 50 µl PCR contained 1X Phusion HF buffer, 200 µM dNTPs, 0.5 µM Forward primer, 0.5 µM reverse primer, 87 ng template DNA and 1-units Phusion DNA Polymerase (NEB, Evry, France). The PCR product was cloned using the hot fusion method (Fu et al., 2014) in pET30a+ vector (Merck, Molsheim France) pre-digested with EcoRI and BglII in order to fusion AgLTP24 with a N-terminal 6XHis flag. Ligation mixture was transformed in Escherichia coli DH5α chemocompetent cells (Chung et al., 1989) for plasmid DNA propagation and sequencing (pET30a-HIS-AgLTP24, supplemental Fig. S1). Plasmids DNA was prepared using (Nucleospin Plasmid DNA kit Macherey Nagel) from 4 independent clones, 2 ml of each E. coli culture grown overnight in 5ml LB medium at 37°C with shaking. Cloned insert DNA was checked by Sanger sequencing (Biofidal, Lyon, France) and used for electrotransformation into iSHuffle T7 lysY E. coli cells (NEB, Evry, France), a strain used to obtain proteins with disulphide bonds (Lobstein et al., 2012) .
5
Molecular Cloning and Protein Engineering
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents and chemicals were obtained from Sigma–Aldrich, except the following: BD (tryptone, yeast extract, TSB powder), Thermo Fisher Scientific (Tris), VWR (glycerol, NaCl, NaNO3), ADM, France (NutriSoy flour), and New England Biolabs (T4 DNA ligase, restriction enzymes). Oligonucleotide primers and two additional synthetic DNA fragments for CPN4 and CPN5 constructs were synthesized by Sigma–Aldrich (Supplementary Data 1 ). The docking domains NDD9 Val and NDD9 Met (Supplementary Table 1 ) were obtained as synthetic peptides from GeneCust. DNA sequencing of PCR products was performed by Sigma–Aldrich and Eurofins.
PCR reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific), or Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) when higher fidelity was required. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin® Gel and PCR Clean-up or NucleoSpin® Plasmid DNA kits (Macherey Nagel, Hoerdt, France).
PCR reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific), or Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) when higher fidelity was required. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin® Gel and PCR Clean-up or NucleoSpin® Plasmid DNA kits (Macherey Nagel, Hoerdt, France).
6
Isotopic Labeling for Biochemical Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biochemicals and media were purchased from VWR (glycerol, NaPi, NaCl, MgSO4), BD (tryptone, yeast extract), Thermo Fischer Scientific (Tris, EDTA), Euromedex (isopropyl β-D -1-thiogalactopyranoside (IPTG)), and Sigma-Aldrich (betaine, imidazole, Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), starch), and Roquette (corn steep). L -proline-2,5,5-D3 and L -serine-2,3,3-D3 were sourced from CDN Isotopes. The enzymes for genetic manipulation were purchased from Thermo Fisher Scientific. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin® Gel and PCR Clean‑up or NucleoSpin® Plasmid DNA kits (Macherey Nagel). Standard PCR reactions were performed with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific); and reactions were carried out on a Mastercycler Pro (Eppendorf). DNA sequencing was carried out by Eurofins.
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!