Genomic DNA from L. lactis DRC3 was isolated from bacteria harvested in the exponential growth phase using Nucleobond® AXG columns and the Nucleobond® buffer set III (Macherey-Nagel Gmbh, Düren, Germany). The protocol used was taken from “Genomic DNA and RNA purification–User manual” of July 2015, revision 08 (Macherey-Nagel GMbh, Düren, Germany) following the “Protocol for Nucleobond® AXG Columns and Nucleobond® Buffer Set III” for “Isolation of genomic DNA from bacteria” with the following modifications. For cell disruption, 30 mg/ml of lysozyme was added, and incubation time was set to 16 h. Incubation at 50°C after the addition of Buffer G4 was set to 1 h, and in the binding step 8 ml of Buffer N2 was used. In the final precipitation step, the obtained pellet was dissolved in 10 mM Tris Buffer (pH 8.00) and incubated at 55°C for 1 h before final storage.
Nucleobond buffer set 3
Manufactured by Macherey-Nagel
Sourced in Germany
The Nucleobond® buffer set III is a collection of buffers designed for the purification of plasmid DNA. It includes buffers for lysis, neutralization, and washing steps, as well as an elution buffer. The set is suitable for both small-scale and large-scale plasmid preparations.
Automatically generated - may contain errors
Lab products found in correlation
Nucleobond axg 100 column, by Macherey-Nagel (2 mentions)
Nucleobond axg column, by Macherey-Nagel (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Nextera xt library preparation kit, by Illumina (1 mentions)
Pcr buffer, by Promega (1 mentions)
Nucleobond axg 20 columns, by Macherey-Nagel (1 mentions)
Hotstart g2 dna polymerase, by Promega (1 mentions)
Sanger sequencing, by Eurofins (1 mentions)
Miseq apparatus, by Illumina (1 mentions)
5 protocols using nucleobond buffer set 3
1
Genomic DNA Isolation from L. lactis DRC3
2
Hybrid Bacterial Genome Sequencing
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For Illumina sequencing, we extracted the bacterial genomic DNA using the standard cetyltrimethylammonium bromide method. The DNA paired-end libraries were prepared using a Nextera XT library preparation kit (Illumina), following the manufacturer’ s protocol and sequenced using the MiSeq Illumina platform and MiSeq reagent kit v.2 (300 cycles; Illumina).
For Nanopore sequencing, genomic DNA was extracted using NucleoBond AXG100 columns and the NucleoBond buffer set III (Macherey-Nagel). Long-read libraries were prepared with the ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies) and sequenced using R9.4.1 flow cells on a GridION platform. The fast5files were base-called using Guppy v.4.0.1 with the settings configdna_r9.4.1_450bps_hac and qscore_filtering.
For Nanopore sequencing, genomic DNA was extracted using NucleoBond AXG100 columns and the NucleoBond buffer set III (Macherey-Nagel). Long-read libraries were prepared with the ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies) and sequenced using R9.4.1 flow cells on a GridION platform. The fast5files were base-called using Guppy v.4.0.1 with the settings configdna_r9.4.1_450bps_hac and qscore_filtering.
3
Genomic DNA Isolation from Bordetella pertussis
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Genomic DNA isolation from Bordetella pertussis
4
Mapping Transposon Insertion Sites in M. hominis
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M. hominis total genomic DNA extractions were performed using 10 mL cultures with NucleoBond® AXG20 columns and the NucleoBond® Buffer Set III from Macherey Nagel according to the manufacturer’s instructions. Transposon insertion sites were then determined by single-primer PCR. The 25 µL final reaction volume contained 1X PCR Buffer (Promega), 3 mM MgCl2, 1 µM of SG9 primer (5′-TTTGGTTCAGAAACTGGTGCT-3′), 0.2 mM dNTPs, 0.1 µL of HotStart G2 DNA polymerase (Promega), and 2.5 µL of transformant DNA. The PCR amplification cycle was performed as previously described53 (link). The insertion positions were determined by Sanger sequencing (Eurofins genomics) of the PCR products with the nested primer MT85-1 (5′-ACAGTAATTGCGGGTGGATC-3′).
5
Genome Sequencing of M. hominis Mutants
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The genomic DNA from four vaa-mutants, two oppA-mutants and from the M. hominis PG21 parental strain was extracted using NucleoBond® AXG100 columns (Macherey-Nagel, Düren, Germany) and the NucleoBond® buffer Set III (Macherey-Nagel). The genomes were sequenced using paired-end V2 2X250 bp sequencing on MiSeq Illumina apparatus (San Diego, CA, USA) after generating the genomic DNA libraries using the of Nextera XT DNA Library Preparation Kit (Illumina). About 160,000 to 200,000 paired reads were obtained for each genome. Data processing including quality check, trimming, alignment with BWA (Galaxy Version 1.2.3) and variant calling using Varscan (Galaxy Version 0.1) was performed using Galaxy at https://usegalaxy.org/ [20 ].
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