All MRSA isolates were cultured on Columbia agar with 5% sheep blood and incubated for 18 h at 37 °C. After incubation, one colony of each strain was transferred to an Eppendorf tube containing 1 mL of tryptic soy broth (Sigma-Aldrich, Darmstadt, Germany) and re-incubated for 18 h at 37 °C. Next, Eppendorf tubes were centrifuged for 5 min at 300× g, and the obtained pellet was washed twice using phosphate-buffered saline (PBS, pH 7.4). Finally, genomic DNA was isolated from the pellet using the GeneMatrix Bacterial and Yeast Genomic DNA Purification Kit (EURx, Gdansk, Poland), according to the manufacturer’s protocol.
Genematrix bacterial and yeast genomic dna purification kit
Manufactured by EURx
Sourced in Poland
The GeneMatrix Bacterial and Yeast Genomic DNA Purification Kit is a laboratory tool designed to extract and purify genomic DNA from bacterial and yeast samples. The kit utilizes a silica-based membrane technology to efficiently capture and concentrate DNA molecules, allowing for high-quality DNA isolation suitable for downstream molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Genematrix basic dna purification kit, by EURx (1 mentions)
C1000 pcr thermal cycler, by Bio-Rad (1 mentions)
Fastdigest, by Thermo Fisher Scientific (1 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Api 50 chl, by bioMérieux (1 mentions)
Pcr thermocycler, by Bio-Rad (1 mentions)
6 protocols using genematrix bacterial and yeast genomic dna purification kit
1
MRSA Genomic DNA Isolation
2
Bacterial DNA Isolation and Genetic Engineering
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Isolation of genomic DNA from K. hansenii ATCC 53582 and plasmid DNA from E. coli TOP 10F bacterial strains as well as DNA purification from agarose gel were performed by solid phase extraction on silica spin columns (Gene MATRIX Bacterial and Yeast Genomic DNA Purification Kit and GeneMatrix Basic DNA Purification Kit, EURx, Gdansk, Poland).
ColorTaq Polymerase (EURx, Gdansk, Poland) and C1000 Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) were used in all PCR amplification procedures (preparation of insert with motAB genes, control over pTI99-motAB vector preparation and verification of K. hansenii transformation) with appropriate primers (Table S1 ) synthesized at Genomed Ltd., Warsaw, Poland.
pTI99-motAB vector preparation was done with standard methods (restriction hydrolysis with BamHI and HindIII enzymes (FastDigest, Thermo Fisher Scientific, Waltham, MA, USA), overnight ligation catalyzed by T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA) and transformation by a heat shock method of competent E. coli TOP 10F cells (prepared in-house).
ColorTaq Polymerase (EURx, Gdansk, Poland) and C1000 Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) were used in all PCR amplification procedures (preparation of insert with motAB genes, control over pTI99-motAB vector preparation and verification of K. hansenii transformation) with appropriate primers (
pTI99-motAB vector preparation was done with standard methods (restriction hydrolysis with BamHI and HindIII enzymes (FastDigest, Thermo Fisher Scientific, Waltham, MA, USA), overnight ligation catalyzed by T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA) and transformation by a heat shock method of competent E. coli TOP 10F cells (prepared in-house).
3
Whole Genome Sequencing of Bacillus subtilis
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The genome of B. subtilis 87Y was isolated using GeneMatrix Bacterial and Yeast Genomic DNA Purification Kit (EurX) and kept at −20 °C. Whole genome sequencing was performed by Eurofins Genomics (Germany) which comprised DNA fragmentation, adapter ligation, size selection, and amplification. The standard genomic library was prepared including unique dual indexing. Sequencing was performed using Illumina technology with pair end run type. Read length was 2 × 150 bp. B. subtilis 168 genome (NCBI: NC_000964.3) was used as a model species.
4
Whole-genome DNA Extraction from E. rhusiopathiae
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Whole-genome DNA was extracted from E. rhusiopathiae strains using the Gene MATRIX Bacterial and Yeast Genomic DNA Purification Kit (Eurx, Gdańsk, Poland) following the manufacturer’s protocol, modified to extend the incubation time of bacteria in the lysis buffer from 15 min to 1 h (gram-positive bacteria, unlike gram-negative bacteria, have a thick cell wall, the removal of which requires a longer incubation of the bacteria with lytic enzymes). DNA concentration was determined using the NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the quality of DNA was checked by agarose (1.5% w/v) gel electrophoresis. The final DNA concentration was ~18 ng/µL.
5
Identification of Lactobacillus plantarum Strain
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The strain AC 11S was identified as L. plantarum by classical phenotypic methods including Gram staining, oxidase (kit HiMedia, Mumbai, India), catalase tests, and a carbohydrate fermentation test with 49 carbon sources (using API 50 CHL, bioMérieux, Marcy l’Etoile, France). The species affiliation was confirmed by Multiplex PCR amplification with primers targeting the recA gene, according to Torriani et al. [17 (link)]. The PCR analysis was performed on a PCR thermocycler (BioRad, laboratories, Inc. group Hercules, CA, USA), using a ready to use PCR mix (IllistraTM PuRe TaqTM Ready To GoTM PCR beads; Amersham Biosciences, Amersham, UK). The target DNA was isolated from an overnight LAB culture of the strain AC 11S using the Gene Matrix Bacterial and Yeast Genomic DNA Purification Kit (EURx Ltd., Gdańsk, Poland), following the manufacturer’s instructions. The PCR reaction conditions were as previously described [18 (link)].
6
16S rRNA Gene Amplification and Sequencing
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Genomic DNA was isolated from purified strains using GeneMatrix Bacterial and Yeast Genomic DNA Purification Kit (EurX) according to the manufacturer's protocol with modified homogenization step (FastPrep-24 bead-beater, one cycle of 20 s at 4.0 m/s). The DNA was analyzed spectrophotometrically (NanoDrop 2000). 16S rRNA gene fragment was amplified using 27F and 1492R primers63 , following the procedure described in Szymańska et al.6 . The products were purified with GeneMatrix PCR/DNA Clean-Up DNA Purification Kit (EurX) according to the manufacturer’s protocol. Sanger sequencing was performed with BrightDye Cycle Sequencing kit (Nimagen), using 40 ng of template DNA, 1.5 pmol of primer and 1 µl of kit and 1.5 µl of BD buffer in 10 µl volume. The reactions were EtOH/NaAc precipitated and read out at IBB PAS, Warsaw, Poland.
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