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7 protocols using chef mammalian genomic dna plug kit

1

Chromosome Size Determination of B. divergens

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PFGE was used to determine chromosome number and size of the B. divergens genome. Asynchronous cultures were centrifuged at 1224 x g for 5 min and washed with RPMI 1640 (Life Technologies Corporation, Carlsbad, CA). The resultant pellets, containing intact cells, were embedded in 0.8% agarose plugs using the clamped homogeneous electric field (CHEF) mammalian genomic DNA plug Kit and treated with proteinase K according to the manufacturer’s instructions (Bio-Rad Labs Inc., Hercules, CA). The CHEF-DR III system (Bio-Rad labs Inc.) was used to separate the intact chromosomes of B. divergens. The PFGE conditions used for a 0.1–2.0 Mb DNA size range were: 6 V cm-1, pulses of 60–120 s for 24 h and an angle of 120°. The conditions used for a 1.8–4.6 Mb DNA size range were: 4.5 V cm-1, pulses of 200 s for 48 h and an angle of 106°. The gels were stained and photographed under ultraviolet transillumination.
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2

Extraction and Analysis of Mferi Genomic DNA

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Mferi cultures
were grown until the pH reached
a value between 7.0 and 7.5. At this point, cultures were supplemented
with chloramphenicol to a final concentration of 100 μg/mL.
The cells were further incubated for 1 h and then harvested by centrifugation,
followed by resuspension in T/S buffer (10 mM Tris pH 6.5, 500 mM
sucrose). The cell suspension was then embedded in 1% low-melting
agarose plugs following the instruction of the CHEF Mammalian Genomic
DNA Plug Kit (Biorad). The quality of the genomic DNA was assessed
by digestion as previously described37 (link) using
50 Units of BssHII restriction enzymes (NEB), followed by PFGE. Prior
to yeast transformation, Mferi genomes were released
by digestion of the agarose matrix with three Units of β-Agarase
I (NEB) per plug. The DNA concentration in the digested plugs was
measured using an Epoch Microplate Spectrophotometer coupled to a
Take3 microvolume plate (BioTek).
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3

Transplantation and Sequencing of Modified Yeast Genome

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Total DNA, including the intact donor genomic DNA from yeast colonies were isolated using a CHEF Mammalian Genomic DNA Plug Kit as per the manufacturer’s instructions (Bio-Rad, Hercules, CA). DNA isolated from yeast cells carrying the Mmc modified genome was transplanted into Mcc recipient cells with polyethylene glycol as described previously
[21 (link), 31 (link)]. The transplanted cells were selected for tetracycline resistance (the tetM gene and the β-galactosidase genes (lacZ) being present on the Mmc chromosome). Mmc genomic DNA containing the Mcc dnaA gene was isolated from the transplants using the BioRobot M48 workstation (Qiagen, Valencia, CA) as per the manufacturer’s instructions. The isolated Mmc genomic DNA from the bacteria transplants was sequenced to confirm the precise, seamless insertion of the Mcc dnaA gene (JCVI Sequencing Facility, MD).
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4

Whole Genomic DNA Transformation in Yeast

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Whole intact Mcap-CAH genomic deoxyribonucleic acid (DNA) was isolated in agarose plugs using the CHEF Mammalian Genomic DNA Plug Kit (Bio-Rad) with some procedural modifications (21 (link)) and was used to transform S. cerevisiae W303a (29 (link)).
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5

Cell Preparation and DNA Extraction for PFGE

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Cells were harvested using trypsin and washed with PBS. Cells were counted using a NC-3000 Advanced Image Cytometer (Chemometec). Each sample was counted twice to increase accuracy. 0.2 × 106 cells were employed to prepare agarose plugs with the CHEF Mammalian Genomic DNA Plug Kit (BioRad, #1703591), according to the manufacturer’s instructions. Pulsed-field gel electrophoresis (PFGE) was performed as previously described49 (link).
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6

Microchromosome DNA Enrichment and Shotgun Sequencing

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Since microchromosomes (MICs) are known to be gene-rich, we specifically prepared MIC-enriched genomic DNA for shotgun sequencing. Blood from PFAM1, diluted with RPMI medium, was separated by centrifugation (400 × g, 30 min) using lymphocyte separation solution (d = 1.077) (Nacalai tesq Co., Japan). Collected blood cells were washed with PBS, and embedded in agarose gel blocks (5 × 107 cells/mL gel). Embedded cells were lysed with detergents and proteinase K using a CHEF Mammalian Genomic DNA Plug Kit (Bio-Rad Labs, Hercule, CA, USA). Agarose blocks containing genomic DNA were loaded directly into wells, and separated by PFGE using a CHEF-DRII apparatus (Bio-Rad) in 0.5% or 0.8% Mega Base Agarose (Bio-Rad) for 72 h in 1xTAE at 14 °C, 2 V/cm. Schizosaccharomyces pombe chromosomal DNA (CHEF DNA Size Marker (Bio-Rad)) was used as a size marker (3.5, 4.6 and 5.7 Mb). The agarose block corresponding to MIC DNA was removed and DNA was extracted from the agarose gel by treating it with thermostable agarase (Nippon Gene Co., Tokyo, Japan) at 60 °C.
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7

High-molecular weight DNA Extraction

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High-molecular weight (HMW) DNA extraction was performed as recommended for the CHEF Mammalian Genomic DNA Plug Kit (BioRad #170-3591). Briefly, cells from the YH or NA12878 cell lines were washed with 2x with PBS and resuspended in cell resuspension buffer, after which 7.5 × 105 cells were embedded in each gel plug. Plugs were incubated with lysis buffer and proteinase K for four hours at 50°C. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to four hours of drop dialysis (Millipore, #VCWP04700) and quantified using Nanodrop 1000 (Thermal Fisher Scientific) and/or the Quant-iT dsDNA Assay Kit (Invitrogen/Molecular Probes).
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