Plasmid DNA and genomic DNA were isolated using the peqGOLD Plasmid Miniprep Kit I and the peqGOLD Bacterial DNA kit (Peqlab Biotechnologie GmbH, Erlangen, Germany), respectively. To improve lysis of M. caseolyticus, cells were first incubated in Solution I of the kit supplemented with 50 mg/l of lysostaphin (Sigma-Aldrich, St Louis, MO, USA) and 2 g/l of lysozyme (Roche Diagnostics, Rotkreuz, Switzerland) for 20 min at 37 °C. For analytical PCR reactions, FIREPol® DNA polymerase (Solis BioDyne, Tartu, Estonia) and GoTaq® Long PCR Master Mix (Promega, Madison, WI, USA) were used for short (<2.5 kb) and long amplicons (up to 20 kb), respectively. Insert amplifications for plasmid construction were performed using High-Fidelity DNA polymerases (Pfu DNA polymerase [Promega] or the Phusion Hot Start II High-Fidelity DNA polymerase [Thermo Fisher Scientific, Waltham, MA, USA]) according to the manufacturer’s instructions. All relevant primers used in this study are listed in Supplementary Table S1 . The presence of mecD was confirmed by PCR using primers mecD-F (5′-TCCTTTAGCGATAGATGGTGAA) and mecD-R (5′-CTCCCATCTTTTCTCCATCCT).
Peqgold plasmid miniprep kit 1
Manufactured by Avantor
Sourced in Germany
The PeqGOLD Plasmid Miniprep Kit is a laboratory equipment designed for the rapid and efficient isolation of plasmid DNA from bacterial cultures. The kit utilizes a silica-based membrane technology to purify plasmid DNA from small-scale preparations.
Automatically generated - may contain errors
Lab products found in correlation
Oligonucleotides, by Eurofins (2 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (2 mentions)
Eporator, by Eppendorf (2 mentions)
Lysozyme, by Roche (1 mentions)
Lysostaphin, by Merck Group (1 mentions)
Pfu dna polymerase, by Promega (1 mentions)
Firepol dna polymerase, by Solis BioDyne (1 mentions)
Peqgold bacterial dna kit, by Avantor (1 mentions)
Gotaq long pcr master mix, by Promega (1 mentions)
Phusion hot start 2 high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
3 protocols using peqgold plasmid miniprep kit 1
1
DNA Extraction and Amplification for M. caseolyticus
2
Electrocompetent Pseudomonas Cells Preparation
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The preparation of electrocompetent Pseudomonas cells was performed according to Choi and Schweizer (2006) (link), and the vectors were introduced by electroporation (2500 V, Eppendorf Eporator®, Hamburg, Germany). DNA manipulation methods and agarose gel electrophoresis were performed as described by Sambrook and Russell (2001) . Enzymes (Phusion High-Fidelity Polymerase, T5 exonuclease, Taq ligase, restriction enzymes, Fast Alkaline Phosphatase) and buffers were purchased from Thermo Scientific Molecular Biology (St. Leon-Rot, Germany) or New England Biolabs (Frankfurt/Main, Germany) and oligonucleotides from Eurofins Genomics (Ebersberg, Germany). Plasmids were isolated using the peqGOLD plasmid Miniprep Kit I from peqLab (Erlangen, Germany) and purified via NucleoSpin Gel and PCR Clean-up from Macherey–Nagel (Düren, Germany) according to supplier protocols. The Gibson Master Mix was prepared according to Gibson et al. (2009) (link). For detailed information, see Supplementary Table S1 and Supplementary Figure S1 .
3
Electrocompetent Cell Preparation and DNA Manipulation
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The used strains and plasmids are listed in Table S1 . E. coli JM101 and P. taiwanensis VLB120 were used for biotransformation studies and E. coli DH5α for cloning purposes. The preparation of electrocompetent Pseudomonas and E. coli cells was performed as described earlier (Sambrook and Russell, 2001 ; Choi and Schweizer, 2006 ), and the vectors were introduced by electroporation (2500 V; Eppendorf Eporator®, Hamburg, Germany). DNA manipulation methods and agarose gel electrophoresis were performed as described by Sambrook and Russell (2001 ). Enzymes (Phusion High‐Fidelity Polymerase, T5 exonuclease, Taq ligase, restriction enzymes, Fast Alkaline Phosphatase) and buffers were purchased from Thermo Scientific Molecular Biology (St. Leon‐Rot, Germany) or New England Biolabs (Frankfurt/Main, Germany) and oligonucleotides from Eurofins Genomics (Ebersberg, Germany). Plasmids were isolated using the peqGOLD Plasmid Miniprep Kit I from peqLab (Erlangen, Germany) and purified via NucleoSpin Gel and PCR Clean‐up from Macherey–Nagel (Düren, Germany) according to supplier protocols. The Gibson Master Mix was prepared according to (Gibson et al., 2009 ). For detailed information, see Supporting Information (Section 1, Table S2 ).
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