sect3
You will require the following:
Tail buffer 50ml
| 1% SDS | 5ml 10% SDS |
| 10mM Tris pH 7.5 | 0.5ml 1M Tris pH 7.5 |
| 50mM EDTA | 5ml 0.5M EDTA |
| 150mM NaCl | 1.5ml 5M NaCl |
| DDW | 38ml |
Phenol/chloroform (1:1 mixture)
0.5M EDTA
4M NH4Ac
Absolute EtOH
70% EtOH
Tris/EDTA pH 7.5
Proteinase K (20mg/ml dissolved in dH2O)
1. This works best with mice that are at weaning age. Take one toe or 1mm of tail and place in a ependorf tube. Don't take more than this, it isn't necessary and too much material interfers further down the track. Also if amount taken are consistant between sample then the amounts used for Southern will be even between samples. If there are a number of samples to be collected, place tubes on ice.
2. Make up a mix of Tail Buffer and Proteinase K allowing 600ul of Tail Buffer with 500ug/ml Proteinase K for each sample (plus an extra dose in case of inaccurate pipetting). Place in a waterbath at 55oC all day, or at least 2hrs.
3. Samples are transferred to hot airshaker overnight at 62oC at a medium-shake speed.
4. To each eppendorf add 500ul of phenol/chloroform (lower phase of mix). Mix by inversion/shake (don't vortex as this shears the DNA). Spin at 13,000 rpm for 2 minutes. Collect supernatant, being careful to avoid the interphase.
5. Place supernatant in fresh eppendorf tube and add:
2ul 0.5M EDTA pH 8.0
200ul 4M NH4Ac
800ul isopropanol
6. Mix by inversion/shake
Spin at 13000 rpm for 5 minutes
Remove and discard supernatant, being extremely careful not to disturb the pellet.
7. To the tube with pellet add 200ul 70% EtOH and vortex
Spin at 13000 rpm for 2 minutes
Remove and discard supernatant (watch the pellet)
8. Add 200ul TE and vortex, allowing DNA to resuspend at room temperature
9. Once DNA has resuspended, digests may be set up. 30ul of DNA/TE suspension per digest should be sufficient, RNAse is not necessary. Digest o/n, ppte with 12ul 5M NaCl, 600ul EtOH and resusp. pellet in 18ul TE and 7ul dye. Allow to dissolve for 20min RT and heat to 37¡C for 5-10 min before loading.