Southern Blotting protocol
Genomic DNA extraction
Cut tails and mark ears: no cuts:1
Left ear cut:2
Right ear cut:3
Both ears cut:4
Place tails in eppendorf with 0.5 ml of lysis buffer with 200 mg/ml of proteinase K
Lysis buffer recipe: 0.1M Tris, pH 8, 5 mM EDTA, 0.2M NaCl. Autoclave and add SDS to give 0.2% (w/v) final concentration. Add proteinase K to 200 mg/ml just before use.
Make up a few spare tubes with lysis buffer.
Make sure tail is submerged.
Digest overnight at 55ºC in shaking waterbath.
Spin eppendorf for 20 min at full speed in microfuge to pellet tail debris
USE WIDE ORIFICE TIPS FROM NOW ON
Take each supernatant trying to avoid hair: should be gelatinous
Place in well of 24 well plate in 0.6 ml of isopropanol and leave to shake on plate shaker for 30-40 min until DNA precipitates become visible.
Transfer DNA to prelabelled eppendorf tube containing 0.5 ml of 70% ethanol.
Spin 10 min at full speed in microfuge.
Aspirate supernatant making sure not to dislodge and lose pellet: make sure pellet is dry.
Add 200-250 ml of TE.
Dissolve DNA: Incubate for 3 hours at 55ºC in non-shaking bath: Resuspend DNA with wide orifice tip and leave overnight to dissolve. Following resuspend DNA with wide orifice tip if necessary. Place in rack (in the right numerical order to keep Heather happy) and store at 4ºC. DO NOT FREEZE GENOMIC DNA.
DIGESTION OF GENOMIC DNA
Use 15 ml of DNA: place in prelabelled eppendorf using wide orifice tip.
Make up digestion mixture: make enough for 4-5 extra tubes.
For IRS-1 per tube you will need
24 ml of water
5 ml of BamH1 enzyme
5 ml of BamH1 buffer
0.5 ml of 100X BSA
For IRS-2
24 ml of H2O
5 ml of Spe1
5 ml of buffer 2
0.5 ml of 100X BSA
Make up the appropriate volume of mix: make sure well mixed and add 35 ml to the 15 ml of DNA in the eppendorf mixing with pipette tip.
Incubate overnight at 37ºC in non-shaking waterbath.
Run gel
Make 0.8% gel
For large gels use 450 ml of TAE and 3.6 g of agarose with 45 ml of EtBr. Make sure gel is cool enough to pour otherwise it will bend the casting apparatus: one large gel has 2x24 well combs and the other has 2x20 well combs and probably takes 400 ml of gel mix
Add 5 ml of DNA dye to the lid of each tube, spin by pulsing in microfuge and load all sample. ALWAYS RUN A POSITIVE HETEROZYGOTE SAMPLE and a 1 Kb ladder.
Run overnight at 30 V or run during day at 120-150 V takes 3-4 hours.
Transfer of DNA
Cut the gel into 4 pieces and photograph gel after it has run: run the lower dye band to with ½ inch of end of gel or beginning of wells for upper set of gels: make sure to label and identify gels and photograph with a ruler.
Place gel in denaturing solution (1.5M NaCl, 0.5M NaOH) for 45 min gently shaking: the upper blue band of the DNA dye will turn green. Gently tip off the denaturing solution and neutralise with 1 M Tris, 1.5M NaCl pH 7.4 for 45-60 min although you can leave longer.
Set up transfer:
Transfer is done with 10 x SSC (1L of 20 x SSC requires 175.3 g of NaCl and 88.2 g of Na citrate pH to 7 with HCl one or two drops is enough.
Cut the lower paper to the exact size of the gel. Cut the two pieces of paper to the same, cut the Hybond N+ nucleic acid membrane to slightly bigger size and cut the wick to slightly narrower although it will need to be long. Wet the Whatman and the membrane and assemble as shown in the picture.
Transfer overnight.
Dissemble the transfer: first take off the wick only and mark the position of the wells on to the membrane.
Cross-link the DNA to the membrane using the UV x-linker in the Kahn lab. Follow the instructions on the machine but have the membranes face up so the side to which the DNA was transferred is up.
Stain for 5 min in water with a little EtBr destain for say 20 min and just check under UV lamp that you have got DNA on the membrane.
Picture of transfer set up.
Hybridization and probe synthesis.
Prehybridization mix:
For 100 mls
30 ml of 20 x SSC
2.5 ml of 20% SDS
50 ml of formamide (kept at 4ºC)
5 mls of 100 x Denhardts (or 10 ml of 50 x)
12.5 ml of H2O (7.5)
Filter the above solution.
While making up this put 1 ml of ds Herring sperm DNA (in –80ºC) to heat on hotblock at 100ºC for 5 min and then transfer to ice for 5 min. Add to the filtered mix above.
Place 50 ml of this prehyb solution on membranes and place in shaking water bath at 42ºC set by thermometer as the thermostat is 2-3ºC out. When prehybing use the white topped tupperware but make sure water does not shake over the edges and make sure that the filters are moving in the box
Prehyb for 4-6 hours.
Meanwhile make probe according to protocol in NEB nick translation kit.
Put plexiglass radiation box in big 37ºC incubator to warm up
Use template at concentration of 100 ng per reaction
Use the Easytide dCTP 6000Ci/mmol. 5 ml
Re-add Klenow after 30 min and let reaction run for 60 min.
Purify probe on G50 column
Remove the top of the column and discard the excess liquid and wash with wash buffer (10 mM Tris pH 7.5, 1mM EDTA)
Remove the bottom cap and run 3 ml of wash buffer through the column
Add the probe to the column
Add 400 ml of the wash buffer and collect this fraction.
Add 400 ml and collect this second fraction: THIS IS THE PROBE.
Count 1 ml of the probe in 6 ml of scintillant it should be between 200000 and 400000 cpm per ml
The second fraction is the probe: if you are going to use it straight away boil it in the hotblock for 5 min and then place on ice for 5 min.
Meanwhile transfer the blots from the white topped tupperware to the high sided good sealing ones and add 20 mls or so of the reminder of the prehyb: make sure that it is clear as the SDS comes out of solution.
Add the probe to the membranes in the 20 mls of prehyb, swirling to make sure that mixing occurs quickly and that the probe does not drop directly onto the membrane.
Hybridize in shaking water bath overnight at 42ºC.
The following morning decant the probe in solution into a clean 50 ml falcon and place in plexiglass box in fridge for reuse.
WHENEVER you REUSE OR ADD PROBE WHICH IS DOUBLE-STRANDED DNA YOU MUST DENATURE BY BOILING FOR 5 MIN THEN PLACING ON ICE FOR 5 MIN BEFORE ADDING TO FILTER. IF YOU ARE BOILING THE WHOLE 20 ML OR SO OF THE PROBE SOLUTION THAT YOU HAVE USED BEFORE YOU NEED TO BOIL IT FOR 15 MIN IN A BEAKER OF BOILING WATER AND THEN ALLOW TO COOL FOR 10-15 MIN ON ICE.
Place the membranes in a fresh tupperware box and wash up the one in which you did the hyb as it is hot.
Wash the membranes twice for 20 min at 42ºC with 2xSSC, 0.5% SDS (made up from 20 x SSC and 20% SDS stocks) and then place on filter paper and get rid of surface wetness and monitor. IF the membrane “sounds” right and this is experience wrap in saran wrap and put down to film at –70ºC overnight in a cassette that does not leak, develop and all things going to plan it should have worked.