Southern blot

Southern blot protocol
Harvest genomic DNA from 50% confluent 100mm plates
Digest 5ul for >1h.
Digest WT DNA in parallel.
Run very pretty gel 100V until blue dye is >2/3 down gel
Take picture with ruler

Denature with denaturing solution 30min
Neutralise with neutralizing solution 30min
(Digest 5ug DNA to make probe)

Transfer
Fill tray with transfer buffer (400ml 10X SSC)
Put glass plate on tray
(Wet everything before assembling. Make sure no air bubbles.)
Hang wick (2 Whatman paper) over glass plate, long enough to drape well into transfer solution
Place gel (well-side down?) on Whatman.
Make sure no air bubbles
Cut Nylon membrane same size as gel (mark membrane), place on gel
Cut Whatman paper same size as gel, place 2 on membrane
Place parafilm around gel to prevent transfer buffer from reaching paper towels
Place 8cm of paper towels on Whatman
Place glass plate and weight on paper towels
Transfer O/N

Immobilize DNA by UV-crosslinking
Put wet membrane + Whatman in Crosslinker. (Optimal cross-linking).
Leave membrane to dry

Prehybridisation
Leave membrane in 10ml prehybridization buffer for 1-2h

Radioactive labelling of probe
Check radioactivity hood for radioactivity
Thaw [a-32P]dATP (use the newest one)
Double glove.
Check on Geiger counter after every step
Use 25-75ng of DNA in 9ul (compare to ladder ~33ng/ band if load 10ul)
Boil DNA 5min to denature
Add 4ul OLB random hexamers when boiling
Plunge in ice
Add 1ul 1mM dGTCTP
In radioactive hood, add 5ul [a-32P]dATP
Add 1ul Klenow (DNA pol I)
Total: 20ul
Mix with pipette tip, quick spin
Use glass plate to shield tube from person when transporting
Incubate at 37C 30min

Purify probe with spin column.
Break off tip.
Open cap.
Spin 1rcf before using to remove liquid and calibrate column.
Add 100ul 6.6.2 to DNA then apply to column.
Spin 1 rcf 5min
Count radioactivity.
Turn off sound.
Should have more than 100 (1-5x 10^6 cpm/ml) (30x 10^6 cpm in total)
Denature probe at >65C 5min

Hybridisation
Add salmon testes in prehyb buffer
Add denatured probe to prehybridisation buffer
Incubate O/N 37C (warm room)

Wash
Rinse with Wash 1
Wash 1 RT 15min
Wash 2 37C 15min x 2
Wash 3 37C 15-30min

Cut film into 2.
Mark corner.
Leave film on membrane, then put intensifier on
-80C O/N
Thaw at 37C for 5-10min.
Develop
Compare with ruler

5x OLB
O: 1.25M Tris-HCl, pH8
125mM MgCl2
A: 1ml solution O
18ul B-ME
B: 2M HEPES, pH6.6
C: Random 6-mer primers in TE at 90 OD/ml
5x OLB: 100ul A
250ul B
150ul C

Hybridization solution (20ml)
DI formamide 10ml
20X SSC 3ml
0.2M EDTA 1ml
1.2M PB 1ml
100X Denhardt's 0.8ml
20% SDS 0.2ml
H2O 5ml

Wash buffers
1: 4X SSC
0.02% SDS
2: 1x SSC
0.5% SDS
3: 0.1X SSC
1% SDS