Mini prep

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 72.06g/200ml	2.5ml
25mM Tris-cl(pH 8.0)	1M stock Tris base 24.22g HCL 8.4ml	2.5ml
10mM EDTA (pH 8.0)	0.5M stock 37.22g/200ml	2ml
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	4ml
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 98.2g/200ml	60.0ml
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