Plasmids were extracted from 14 samples of hematologic malignancy individuals, including both ESBL-positive and ESBL-negative isolates. In addition, 17 random samples collected from non-hematologic malignant individuals were chosen to be extracted according to the type of the sample. Plasmid extraction was performed using Promega PureYieldTM Plasmid Miniprep System (Catalogue #A1223, USA) and according to the manufacturer’s instructions. After plasmid extraction, the concentration of plasmid from each isolate was measured using a NanoDrop™ 1000 Spectrophotometer (Thermofisher Scientific, Wilmington, USA). 1.5 µl DNA was needed to measure plasmid concentration at wavelength of 260 nm. An appropriate volume of 10 µl of the extracted plasmid DNA obtained from hematological malignancy patient isolates was loaded after mixing with loading dye (KAPA BIOSYSTEMS, USA). Plasmids were electrophoresed through 0.8% agarose gel under 100 V for 2 h. Plasmid bands were visualized with Ethidium bromide staining under UV transilluminator (Biometra, Germany). Sizes of the bands were compared with the lambda HindIII digest ladder (New England, BioLabs).
Loading dye
Manufactured by Roche
Sourced in United States
Loading dye is a chemical solution used in molecular biology and biochemistry. It is primarily used to visualize and track the migration of DNA or RNA samples during gel electrophoresis. The dye helps to increase the density of the samples, allowing them to sink into the wells of the gel. It also contains a colored component that migrates through the gel, indicating the progress of the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pureyieldtm plasmid miniprep system, by Promega (1 mentions)
Uv transilluminator, by Analytik Jena (1 mentions)
Kapa2g fast hotstart dna polymerase, by Roche (1 mentions)
Tri reagent, by Merck Group (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Reverse transcriptase, by New England Biolabs (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Hoechst 3342, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Merck Group (1 mentions)
Osteo assay plate, by Corning (1 mentions)
Dntps, by Roche (1 mentions)
Foetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
3 protocols using loading dye
1
Plasmid Extraction and Characterization
2
RNA Extraction and RT-PCR Analysis
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Matched normal and tumor samples were obtained from the Hadassah Medical Center tissue bank, under institutional Helsinki ethical approval. RNA was extracted from the frozen tissues using metal beads in TRI reagent (Sigma) in a bullet blender at 4°C for 5 min. Total RNA (500 ng) was reverse transcribed using M-MLV reverse transcriptase (Promega). PCR was performed on 1/50 (2 μl) of the cDNA, in 25 μl reactions containing KAPA2G Fast HotStart DNA Polymerase in a ×2 ReadyMix format with loading dye (Kapa Biosystems), 0.2 mM of each primer and 8% (v/v) DMSO. PCR conditions were 95°C for 5 min, then 34 cycles of 94°C for 15 s, 60°C for 15 s and 72°C for 45 s, followed by 10 min at 72°C. PCR products were separated on 1.5% or 2% agarose gels. Normalization was performed using β-actin. Primers are listed in Supplementary Table S11.
3
Optimized Cell Culture Techniques for Bone Research
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Trypan blue, crystal violet, antibiotic-solution containing: streptomycin, fungizone and penicillin, and primary antibodies for western blotting were purchased from Sigma-Aldrich (St Louis, MO, USA).
Dulbecco's minimum essential medium (DMEM) was provided by Sterilab Services (Harrogate, UK) and foetal bovine serum (FBS) was supplied by Biochrom (Berlin, Germany). RANKL was acquired from Research and Diagnostic Systems (R&D Systems, Minneapolis, MN, USA). Cell cluster plates and glass cover slips were purchased from LASEC (Cape Town, South Africa (SA)). Osteoassay plates were purchased from Corning inc. (Corning, NY, USA). Fermented rooibos tea (red rooibos) was obtained from National Brands Limited (Rivonia, Durban, SA). Unfermented rooibos tea (green rooibos) was obtained from Biedouw Valley (Clanwilliam, Western Cape, SA). Phalloidin-conjugate and Hoechst 3342 were purchased from Life Technologies (Carlsbad, CA, USA). IκB and GAPDH rabbit polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Secondary antibodies for western blotting were purchased from Bio-Rad (Hercules, CA, USA). Reverse transcriptase was purchased from New England Biolabs (Ipswich, MA, USA). Oligo (dT) primers were purchased through Thermo Scientific (Waltham, MA, USA). Other PCR reagents (dNTPs, DNA polymerase, DNA ladder, loading dye, etc.) were supplied by KAPA Biosystems (Cape Town, SA).
Dulbecco's minimum essential medium (DMEM) was provided by Sterilab Services (Harrogate, UK) and foetal bovine serum (FBS) was supplied by Biochrom (Berlin, Germany). RANKL was acquired from Research and Diagnostic Systems (R&D Systems, Minneapolis, MN, USA). Cell cluster plates and glass cover slips were purchased from LASEC (Cape Town, South Africa (SA)). Osteoassay plates were purchased from Corning inc. (Corning, NY, USA). Fermented rooibos tea (red rooibos) was obtained from National Brands Limited (Rivonia, Durban, SA). Unfermented rooibos tea (green rooibos) was obtained from Biedouw Valley (Clanwilliam, Western Cape, SA). Phalloidin-conjugate and Hoechst 3342 were purchased from Life Technologies (Carlsbad, CA, USA). IκB and GAPDH rabbit polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Secondary antibodies for western blotting were purchased from Bio-Rad (Hercules, CA, USA). Reverse transcriptase was purchased from New England Biolabs (Ipswich, MA, USA). Oligo (dT) primers were purchased through Thermo Scientific (Waltham, MA, USA). Other PCR reagents (dNTPs, DNA polymerase, DNA ladder, loading dye, etc.) were supplied by KAPA Biosystems (Cape Town, SA).
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