LECTURE 10 - SOUTHERN BLOTTING

what is the purpose of southern blotting

identify a part of DNA containing the specific gene of interest

what are the 3 principal steps of a southern blot

1. separate DNA on agarose gel

2. treat DNA, breaking it into fragments small enough to be passed out of gel

3. blotting of gel to allow movement of fragments

what percent agarose gel is used

0.7 - 1%

what solutions are used to treat electrophoresed DNA, and explain what each does

0.25M HCl → partial depurination

0.4M NaOH/0.6M NaCl → breaks carbohydrate backbone at apurinic sites

what are the 3 methods used to transfer DNA from gel to a solid matrix

capillary blotting

vacuum blotting

electroblotting

what are the 2 types of membranes which can be used

nitrocellulose

nylon based

describe nitrocellulose membrane strength

very fragile

describe nylon based membrane strength

very strong

how is DNA bound to a nitrocellulose membrane

by baking it at 80*C

how is DNA bound to a nylon based membrane

by drying or UV cross linking

explain how capillary blotting works

the blotting solution is drawn up by filter paper

the solution rises through the hybridisation membrane and paper towel stack

DNA is carried up to the membrane with the fluid

what are the pros and cons of capillary blotting

pros; cheap

cons; time consuming and can result in poor resolution

explain how vacuum blotting work

a vacuum pulls the buffer/DNA down through the gel and membrane

what are the pros and cons of vacuum blotting

pros; only needs brief treatment of DNA, quicker, good resolution

cons; specific apparatus required,

explain how electroblotting works

uses electrophoresis to transfer the DNA

a gel with separated DNA has positive and negative electrodes applied

the membrane is situated between the gel and the positive electrode

upon applying voltage the DNA moves towards the positive electrode, hitting the membrane in the process

what are the pros and cons of electroblotting

pros; needs no treatment to fragment DNA, very quick,

cons; quite expensive, resolution depends on set up

what membrane does electroblotting use

nitrocellulose