Genomic library

 

Purpose

Investigation of entire structures of genes and to study regulation of gene expression. This is also essential for genetic transformation.

 

Preparation

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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