Investigation of entire structures of genes and to study regulation of gene expression. This is also essential for genetic transformation.
1 Isolation of nuclei from cells (when nuclear encoded genes are targeted) and purification of them--- Using differential centrifugation technique contaminations of genomes derived from mitochondira and plastids.
2 Isolation of a large molecular weight genomic DNA from the nuclei.
3 Fractionation of digested DNA by restriction enzymes using a gradient centrifugation in a sucrose density gradient.
An example of fractionation of pea genomic DNA fragments digested with EcoR1.
In the above fractionation, fragments appeared in the fraction numbers 4 and 5 (4 to 10 kbp) were ligated into a vector. Nuclei were purified from 5 day old pea stems grown in the dark.
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