Chef mammalian genomic dna plug kit
The CHEF Mammalian Genomic DNA Plug Kit is a laboratory tool designed for the preparation of high-molecular-weight genomic DNA from mammalian cells. It provides a method for encapsulating cells in agarose plugs, which protects the DNA from shearing during the extraction process.
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7 protocols using chef mammalian genomic dna plug kit
Chromosome Size Determination of B. divergens
Extraction and Analysis of Mferi Genomic DNA
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).
Transplantation and Sequencing of Modified Yeast Genome
[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).
Whole Genomic DNA Transformation in Yeast
Cell Preparation and DNA Extraction for PFGE
Microchromosome DNA Enrichment and Shotgun Sequencing
High-molecular weight DNA Extraction
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