Southern blots – E.M. Southern (1975)

– analysis of DNA by transfer to membrane

– inspiration for: Northern blot (RNA)

Western blot (protein)

– found nitrocellulose would bind denatured DNA

– probe for sequences homologous to labelled probe

– H-bonding between single-stranded nucleic acids

advantages: – efficiently binds nucleic acids

– either side of membrane can be used

disadvantages: – fragile – tears easily

– sensitive to alkaline conditions

– expensive

– very flammable (“smokeless powder”)

nylon membranes – alternative to nitrocellulose

advantages: – durable – do not tear easily

– resistant to alkaline conditions

– efficiently binds nucleic acids

– binding is covalent

– hybridized probes can be “stripped” from blot

– high temperature/low salt wash

– covalently bound blot DNA remains on nylon

disadvantages: – expensive

– many types only bind nucleic acids on one side

– must put right side down for DNA binding

(Hybond membranes 2-sided)

“unblot” protocol

– dry gel onto plastic support (gel dryer)

– probe dried gel directly

advantages: – cheaper than membranes

– no prehybridization needed before probing

disadvantages – large probes do not enter gel efficiently

(e.g. denatured plasmid DNA)

– probes cannot be “stripped”

protocol:

1. digest DNA sample, size separate on agarose gel

2. soak gel in acid (causes partial depurination)

– depurinated sites cleaved in alkaline solution

– breaks DNA into smaller fragments

(fragments > 1 kb long not transferred efficiently)

3. soak in alkaline solution – denatures DNA

4. neutralize in Tris/NaCl solution

– optional for nylon blots

5. transfer DNA to membrane

capillary transfer – traditional method

– membrane sandwiched between gel and stack of paper

– liquid in gel absorbed by paper – DNA carried along

– sticks to membrane

– may be done using only liquid in gel (lab 4 protocol)

– or suspend sandwich above tank of neutralization buffer

– paper wick connecting tank and sandwich (Fig. 1.8)

– longer transfer time

– get more complete transfer

– better for small amounts DNA

electroblotting – alternate procedure

– need special apparatus

– uses electrophoresis to transfer DNA fragments

– larger fragments transferred

6. if using nitrocellulose – bake 2 hours at 80°C

– fixes DNA to filter – not needed for nylon