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Southern Analysis of Mouse Toe / Tail DNA

来源:生物谷  2010/7/2   访问量:2095
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You will require the following:

- Tail buffer 50ml
10% SDS 5ml
1M Tris pH 7.5 0.5ml 
0.5M EDTA 5ml 
5M NaCl 1.5ml
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 interferes further down the track. Also if amount taken are consistent 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).
  3. Place in a waterbath at 55ºC overnight.
  4. To each ependorf 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 ependorf 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. Add 200ul TE and vortex. Place samples at 65 degrees for 5 min. with caps open. Allow DNA to resuspend at room temperature.
  8. 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 resuspend pellet in 20ul TE. Place at 65 degrees for 5 min. with caps open. Allow samples to sit at room temperature for about 20-30 minutes before loading.

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