Mini prep

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Plasmid DNA mini-prep (Manual methods)


Reagent Set up

Alkaline lysis solution 1 (4°C store)

Component Stock(200ml) Working(100ml)
50mM Glucose 2M stock
25mM Tris-cl(pH 8.0) 1M stock
Tris base 24.22g
HCL 8.4ml
10mM EDTA (pH 8.0) 0.5M stock
D.W 193ml
Total volume 200ml

Alkaline lysis solution 2 (store at RT)

Component Stock(200ml) Working(100ml)
0.2N NaOH 10N stock
80g/160ml D.W
1% w/v SDS SDS powder 2g
D.W 196ml
Total volume 200ml

Alkaline lysis solution 3 (Store 4°C)

Component Stock(200ml) Working(100ml)
5M Potassium acetate 5M stock
Glacial acetic acid 100% 11.5ml
D.W 28.5ml
Total volume 100ml

Reaction Protocol

  1. Prepare samples (picked single cell with media/1.5 or 2ml tube, 16h incubated)
  2. Media tube centrifuge spin down 1min
  3. Solution 1. Add 200ul voltexing
  4. Solution 2. Add 200ul gently inversion 15sec - 실처럼 엉기는 것을 확인
  5. Solution 3. Add 200ul inversion
  6. Centrifuge 12000rpm, 10min at RT
  7. New 1.5ml tube, labeling
  8. Transfer upper phase and same volume of Isopropanol(DNA down)
  9. 500ul 70%EtOH, pellet rinse
  10. 12,000rpm, 5min at 4°C or RT
  11. Dry EtOH and add D.W 20ul