Modified Pulsed Field Gel Electrophoresis (PFGE) Protocol for typing K.pneumoniae was used as described by Zakaria and Hassuna (2019) (link). Briefly, 24 K. pneumoniae isolates were recovered from −70°C stock culture and streaked on Mueller-Hinton agar (Oxoid, United States). All Culture plates were incubated at 37°C for confluent growth without exceeding a 24-h incubation. Plugs were made by adding an equal volume of molten 1% SeaKem Gold (SKG, Lonza, United States) agarose to the cell suspension, and the mixture was immediately dispensed into the wells of a disposable plug mold (Bio-Rad Laboratories, United States). Plugs were carefully transferred from the mold into new sterile 5 ml plain tubes containing 2 ml lysis buffer. Tubes were incubated overnight at 56°C in shaker incubator with vigorous agitation (170–180) rpm. Plugs were restricted with 50 U Xba1 enzyme (New England Biolabs, United States) and 2 μl BSA (10 mg/ml Thermo Fisher Scientific) and incubated for 16 h at 37°C. Digested slices were loaded into 1% seakem gold agarose with electrophoresis conditions: initial switch time 6.7 s and final switch time 35.6 s and 6 V for 19 h at 14°C. Lambda ladder (New England Biolabs) was used as a molecular size marker.
Lambda ladder
Manufactured by New England Biolabs
Sourced in United Kingdom
The Lambda Ladder is a DNA molecular weight standard used for size estimation of linear double-stranded DNA molecules in agarose gel electrophoresis. It consists of DNA fragments ranging in size from 48.5 to 1,018 kilobase pairs, derived from lambda phage DNA.
Automatically generated - may contain errors
Lab products found in correlation
Chef dr 3 system, by Bio-Rad (2 mentions)
Disposable plug mold, by Bio-Rad (1 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (1 mentions)
Xbai, by New England Biolabs (1 mentions)
Chef mapper xa system, by Bio-Rad (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
4 protocols using lambda ladder
1
Molecular Typing of K. pneumoniae using Modified PFGE
2
Pulsed-Field Gel Electrophoresis of Chromosomal DNA
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Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with XbaI (New England BioLabs, MA, USA) was performed according to a standard protocol [21 (link)]. Electrophoresis of the prepared samples was performed on the CHEF-DRIII system (Bio-Rad Laboratories) with 3 litres of 0.5X TBE running buffer. The gels were run with an initial switch time of 2.2 sec, a final switch time of 54.2 sec, a run time of 22 hours, an angle of 120°, a gradient of 6.0 V/cm, and a temperature of 14°C. Lambda ladder (New England BioLabs) was used as a molecular weight marker. Each gel was stained with 1 μg/ml ethidium bromide for 30 minutes and destained with distilled water for 30 minutes. The gels were photographed under UV transillumination. DNA fingerprints were analysed using BioNumerics Software (Applied Maths, Keistraat sint-Martens-laten, Belgium). Cluster analysis was performed based on the Dice coefficient with a 1.8% band tolerance and an optimisation of 4% for comparisons of DNA profiles. A similarity coefficient of 80% was applied to the generated dendrogram, which corresponds to the criteria of Tenover et al. [22 (link)].
3
Chloroplast DNA Extraction and Analysis
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The chloroplasts suspended in CIB were mixed with 1% low-melting point agarose (Promega, V2111) dissolved in TE buffer (1:1, v/v) at 37 °C. The plugs were solidified at 4 °C for 30 min and then lysed in lysis buffer (1% sarkosyl, 0.45 M EDTA, 10 mM Tris-HCl (pH 8.0) and 2 mg/mL proteinase K) at 48 °C for 16 h with shaking. The lysis buffer was exchanged three times. Agarose plugs were then washed in 1x TE buffer 6 times at 4 °C, with the first two washes containing 1 mM PMSF, filled into 1% agarose gel, and subjected to electrophoresis in 0.5x TBE for 42 h at 14 °C using a CHEF Mapper XA system (Bio-Rad). A Lambda Ladder (New England Biolabs; N0341) was used to indicate the molecular weight. The detailed electrophoresis parameters were 5 to 120 s of pulse time at 4.5 V/cm. After EtdBr staining and photography, the gel was blotted onto a Hybond N+ membrane (GE, RPN303B) according to standard DNA gel blotting methods. A 505-bp fragment of the chloroplast rbcL gene (55677–56181) was labeled with [α-32P]-dCTP (NEG513H) using Random Primer DNA Labeling Kit Ver. 2 (Takara, 6045) and used as a probe for hybridization. The autoradiographs were exposed to the phosphor screens for 5 days and then scanned using Typhoon FLA9500.
4
Pulsed-field Gel Electrophoresis Analysis
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The clonal relationship among the 130 isolates was revealed by comparison of SmaI digested DNA profiles, using PFGE analysis. Highmolecular-weight DNA was isolated from 1 mL of an overnight culture, as previously reported (Novais et al., 2004; Tenover et al., 1995) . The digested plugs were subjected to electrophoresis, using the CHEF-DR III system (Bio-Rad Laboratories, Hercules, CA, USA), in a 1.2% agarose gels at 6 V/cm, with linear switching interval ramps from 35 s to 25 s for 25 h at 14 °C for the first block. Lambda ladder (New England Bio-Labs, Beverly, MA, UK) was run as molecular weight marker. After staining with gel red (Biotium), DNA bands were visualized by UV. The images acquisition was performed by using ChemiDoc™ MP Imaging System (Bio-Rad Laboratories Inc. Hercules, California, USA). The PFGE profiles were converted to TIFF files and subjected to cluster analysis, using BioNumerics v. 7.5 software (Applied Maths, Sint-Martens-Latem, Belgium). PFGE profiles were interpreted according to criteria of Tenover et al. (1995) . PFGE band patterns were generated by BioNumerics v. 7.5 software (Applied Maths, Sint-Martens-Latem, Belgium) with tolerance position of 1%. Clustering was based on the unweighted pair group method with arithmetic averages (UPGMA). The Dice correlation coefficient was used to detect the similarities of banding patterns.
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