Single CD34+ cell-derived CFUs were grown for 14 days in MethoCult™ H4435 Enriched (STEMCELL Technologies). For infusion product CFUs, CD34+ cells electroporated with Cas9 RNP were incubated for 24 hours at 32°C, then plated with 1.1ml of MethoCult H4435 in 35-mm CFU dishes at a density of 2000 cells/dish. For ZL33’s 4.5m BM CFUs, 20 ml of BM aspirate was collected, and CD34+ cells were immunoselected as previously described (Donahue et al., 2005 ; Wu et al., 2014 (link)). BM-derived CD34+ cells were plated with 1.1ml of MethoCult H4435 in 35-mm CFU dishes at a density of 2000 cells/dish. The remaining cells were used for targeted deep sequencing. Single CFU colonies were picked, and DNA was extracted with the Maxwell® 16 Cell LEV DNA Purification Kit (Promega, AS1140) on day 14 of plating.
Maxwell 16 cell lev dna purification kit
Manufactured by Promega
Sourced in United States
The Maxwell® 16 Cell LEV DNA Purification Kit is a laboratory equipment product designed for the automated extraction and purification of DNA from various biological samples. It utilizes magnetic bead technology to efficiently capture and isolate DNA, providing a reliable and consistent method for DNA isolation.
Automatically generated - may contain errors
Lab products found in correlation
Quant it high sensitivity dna assay kit, by Thermo Fisher Scientific (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Methocult h4435 enriched, by STEMCELL (1 mentions)
Quantstudio 12k real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman cnv assay, by Thermo Fisher Scientific (1 mentions)
Vacutainer cpt, by BD (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Na18972, by Coriell Institute for Medical Research (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Abi bigdye terminator kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
K2edta tubes, by BD (1 mentions)
Tempus blood rna tube, by Thermo Fisher Scientific (1 mentions)
Preserved blood rna purification kit 1, by Norgen Biotek (1 mentions)
Bioanalyzer 6000 nano kit, by Agilent Technologies (1 mentions)
Globin zero gold rrna removal kit, by Illumina (1 mentions)
Qubit rna hs, by Thermo Fisher Scientific (1 mentions)
14 protocols using maxwell 16 cell lev dna purification kit
1
Clonogenic Assay of Hematopoietic Cells
2
Measurement of AMY1 Copy Numbers
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AMY1 copy numbers were measured in biobanked samples of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated by centrifuging fasting whole blood samples collected in BD Vacutainer® CPT™ cell preparation tubes with sodium citrate. The harvested PBMC pellet was resuspended in fetal bovine serum (FBS) with 10% dimethylsulphoxide (DMSO) and stored at −80 °C until analysis. Genomic DNA from PBMCs was isolated using the Maxwell 16 Cell LEV DNA purification kit (Promega, Fitchburg, WI, USA), and its purity and concentration was assessed using a NanoDrop One spectrophotometer (Thermofisher, Waltham, MA, USA). AMY1 gene copy numbers were estimated by duplex quantitative real-time PCR (2qPCR) on a Life Technologies QuantStudio™ 12K Real-Time PCR system, with QuantStudio™ software version 1.2.2, with a protocol adapted from Falchi et al. [9 (link)], and analysed with CopyCallerR software version 2.1 (Thermo Fisher Scientific, Waltham, MA, USA). Each sample reaction consisted of two TaqMan CNV assays (Life Technologies, Carlsbad, CA, USA); one specific for the target, AMY1 (Hs07226362_cn), and one specific for the reference gene (RNase P). Each sample was run in quadruplicate, and each run included an externally validated 14-copy control (NA18972, Coriell Cell Repositories, Camden, NJ, USA) along with a negative control.
3
Nextera XT DNA Sequencing Protocol
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For the GeneSippr analysis, isolates (Table 2 ) were cultured on nutrient agar (Difco, Becton, Dickinson & Co) overnight (14–16 hrs) at 37°C, and genomic DNA was extracted from single colonies using the Maxwell 16 Cell LEV DNA Purification kit (Promega, Madison, WI). DNA was quantified using the Quant-iT High-Sensitivity DNA Assay Kit (Life Technologies Inc., Burlington, ON). Sequencing libraries were constructed from 1 ng of gDNA using the Nextera XT DNA sample preparation kit (Illumina, Inc., San Diego, CA) and the Nextera XT index kit (Illumina, Inc.). Genomic sequencing of eight multiplexed samples was performed on the Illumina MiSeq Platform (Illumina, Inc.) using a 300 cycle MiSeq Reagent kit v2 or a 600 cycle MiSeq Reagent kit v3 (Illumina, Inc.). Paired-end sequencing was conducted with 21 base reads generated from the first strand and 281 (300 cycle kit) or 581 (600 cycle kit) base reads from the second strand.
4
Genomic DNA Purification and TSC2 Sequencing
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Total genomic DNA was purified from primary cells using Maxwell®16 Cell LEV DNA Purification Kit (Promega, Italy), according to the manufacturer’s instructions.
Direct sequencing by Sanger method of TSC2 exons 1–42 and exon-intron splicing junction boundaries was performed as routinely by the Laboratory of Molecular Genetics.
PCR reactions were treated with exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT, USP Corporation, Ohio, USA) and sequence reactions were performed by ABI BigDye® Terminator kit (Applied Biosystems, Foster City, CA, USA) and analysed by ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequence data were analyzed using Mutation surveyor DNA variant analysis software (Softgenetic PA, USA).
Direct sequencing by Sanger method of TSC2 exons 1–42 and exon-intron splicing junction boundaries was performed as routinely by the Laboratory of Molecular Genetics.
PCR reactions were treated with exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT, USP Corporation, Ohio, USA) and sequence reactions were performed by ABI BigDye® Terminator kit (Applied Biosystems, Foster City, CA, USA) and analysed by ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequence data were analyzed using Mutation surveyor DNA variant analysis software (Softgenetic PA, USA).
5
Multiomics profiling of peripheral blood
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Peripheral blood for gene expression and DNA methylation analyses was sampled simultaneously in Tempus Blood RNA Tubes (Thermo Fisher Scientific, Waltham, MA, USA) and K2-EDTA tubes (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA), respectively. Total RNA was isolated from the Tempus Blood RNA Tubes using the Preserved Blood RNA Purification Kit I (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions. DNAse treatment was carried out as recommended. The quality and concentration of the RNA were measured using the BioAnalyzer 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA) and Qubit RNA HS (Thermo Fisher Scientific), with a mean RNA integrity number (RIN) of 9.2 and a concentration of 159 ng/μL. The total RNA samples were depleted for ribosomal RNA and globin transcripts with the Globin-Zero® Gold rRNA Removal Kit (Illumina, San Diego, CA, USA).
DNA was isolated from the K2-EDTA tubes using the Maxwell 16 Cell LEV DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The quality and concentration of the DNA were measured using Nanodrop (Thermo Fisher Scientific). Bisulfite conversion of DNA (500 ng) was done using the EZ-96 DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.
DNA was isolated from the K2-EDTA tubes using the Maxwell 16 Cell LEV DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The quality and concentration of the DNA were measured using Nanodrop (Thermo Fisher Scientific). Bisulfite conversion of DNA (500 ng) was done using the EZ-96 DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.
6
Automated DNA Purification with Maxwell
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The Maxwell® 16 Cell LEV DNA Purification Kit (Promega, Madison, Wisconsin, USA) was used with the automated Maxwell® 16 Instrument (Promega) configured with the low elution volume (LEV) hardware according to the manufacturer’s instructions. Samples (100 µL) was subjected to the purification kit together with 100 µL elution buffer.
7
DNA Extraction from Fixative-Treated Samples
8
Diagnostic PCR for Clone Identification
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All PCR reactions were performed using Taq DNA polymerase (Ecogen, Madrid, Spain) in a 25 μL reaction mixture containing 5 μL of DNA as a template, following the manufacturer’s recommendations. For diagnostic PCRs, DNA from single clones was extracted using the Maxwell® 16 Cell LEV DNA Purification Kit (Promega, Madison, WI, USA). A schematic representation of the diagnostic PCRs and primers used can be found in Figure 1 A,B and Table 1 .
9
Targeted Genomic DNA Sequencing
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Genomic DNA was isolated by Gentra Puregene Cell Kit (Qiagen, Hilden, Germany) or Maxwell 16 Cell LEV DNA Purification Kit (Promega, Madison, WI, USA) from bone marrow mononuclear cells obtained by Ficoll gradient (Cedarlane Laboratories Ltd., Burlington, ON, Canada). For library preparation, we fragmented 600 ng–1 µg of genomic DNA to an average size of 600 bp by Covaris S220 Focused-Ultrasonicator (Covaris, Woburn, MA, USA) or Kapa enzymes (Kapa Biosystems, Wilmington, MA, USA). Libraries in the pre-hybridization steps were prepared with the KAPA HyperPlus Kit (Kapa Biosystems, Wilmington, MA, USA) and were then hybridized to our custom-designed EZ SeqCap gene panel (Roche NimbleGen, Madison, WI, USA). The pool of enriched libraries was sequenced on a MiSeq platform, using 300 bp paired-end reads (v3 chemistry, Illumina, San Diego, CA, USA).
10
Isolation and Characterization of CTCs
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DNA from the primitive lesions in paraffin embedded tumors was extracted using the Maxwell16 FFPE plus LEV DNA purification Kit (Promega, Madison, USA). For CTC isolation, we followed the protocols described as the previous paper (25 (link)). Briefly, 5 mL peripheral blood from the patient’s vein was first lysed using red blood lysis buffer and then filtered through an 8 µM filter membrane. CTCs remained on the filter membrane, whereas white blood cells passed through the filter, after which, their DNA was extracted using the Maxwell 16 cell LEV DNA Purification kit (Promega, Madison, USA). The membrane with CTCs was hybridized using CanPatrolTM CTC RNAISH (SurExam Bio-Tech, Guangzhou, China) and identified. Then, confirmed CTCs were collected using a Palm Microbeam laser microdissection system and further enriched and amplified with the GenomePlex Single Cell. CTCs were classified as epithelial, mesenchymal, and mixed types according to their morphology and surface markers. Their graphs were shown as Figure 1 . Whole Genome Amplification Kit (Sigma, USA). Finally, DNA fragments were obtained from amply and purification and were used for all subsequent experiments.
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