DNA - Restriction Enzyme Digestion and Gel Electrophoresis

commercially prepared restriction enzyme, Eco RI, to do what?

-to digest the samples of DNA we isolated last week. This enzyme will cleave double stranded DNA whenever it encoun¬ters the sequence 5'-GAATTC-3'.Digesting complex DNA, like the Didymium iridis genome, will generate many fragments of all dif¬ferent sizes

what happens when digested DNA is loaded into wells in a gel matrix and exposed to an electrical current?

-the negatively charged DNA fragments will migrate towards the positive electrode (this technique is called gel electrophoresis). Smaller DNA fragments will move through the matrix more rapidly than larger fragments. We can expect to see a nearly continuous series of DNA bands on our gel, each band representing many copies of each size fragment.

in order to see DNA in the gel what must be done?

-In order to see the DNA in the gel, it must be stained with the dye ethidium bromide (EtBr) and viewed with an ultraviolet light source

During the DNA isolation procedure chromosomal DNA becomes sheared?

-at random positions, if due care is taken during DNA isolation the resulting DNA fragments are still quite large (>50kb). Fragments of this size will migrate very slowly in an agarose gel matrix

Careful isolation technique will result in a?

-single high molecular weight band, representing all fragments above about 20kb (all the fragments that size and above migrate at the same rate).

DNA which has been handled roughly will have?

-some high molecular weight fragments and a proportion of molecules below 20 kb which will appear as a smear of different sized fragments below the 20 kb band.

DNA concentration after resuspension can be checked by several methods?

1) by spotting a 1µl sample onto agarose with 0.5 µgm/ml EtBr and observing the intensity of fluorescence of the spot under UV illumination (we will have an example in lab), 2) by running a gel, 3) by measuring the DNA sample absorbance at 260 nm (UV spectrophotometer).