実験関係ノート #2 NID-miniprep

NID miniprep
長所：チューブ1本でminiprepできる
　　　カラムいらない
短所：カラムよりはちょっと汚い（制限酵素処理には使える）
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023457

1) 1.5-2 ml of bacterial cultures were pelleted at 6000-7000 rpm for 1 min.
2) After drawing 150 µl extraction buffer into a pipette tip, the pellet was loosened off the tube wall with the tip without releasing the buffer. Then the extraction buffer was added and the pellet resuspended.
3) The bacterial suspension was incubated at 65°C for 5 min.
4) Suspensions were centrifuged at maximum rpm for 10 min or until a tight bacterial pellet was formed. The pellet was removed with a toothpick.
5) 100-120 µl isopropanol was added, followed by mixing and centrifugation of the solution at 7000 rpm for 10 min at RT.
6) DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions. After discarding the supernatant, the DNA was centrifuged after adding 70% ethanol. Ethanol was removed, and the DNA pellet was dissolved in 20-50 µl TE buffer.

The composition of the extraction buffer was: 5% sucrose, 20–50 mM EDTA, 50 mM Tris pH 8, 0.75 M NH4Cl, 0.5% IGEPAL CA-630 (or Triton X-100), lysozyme 100 µg/ml, and RNase A 25 µg/ml. Addition of 20–50 mM CaCl2 to the extraction buffer reduces extraction of chromosomal DNA and large plasmids, but greatly facilitates formation of cellular debris during sedimentation. A 100x enzyme stock containing 10 mg/ml lysozyme and 2.5 mg/ml of RNase A prepared in 50% glycerol and 50 mM Tris pH 8 was stored at −20°C and used repeatedly.

実際の手順
NID-buffer (100ml)
 Sucrose 5g (final. 5%)
 0.5M EDTA 10ml (final. 50mM)
 2M Tris-HCl pH8.0 2.5ml (final. 50mM)
 NH4Cl 4g (final. 0.75M)
 Triton-X100 500μl (final. 0.5%)
 Lysozyme 10mg (final. 100μg/ml)
 100mg/ml RNase A 25μl (final. 25μg/ml)
 蒸留水で100mlに, 滅菌不要, 室温で保存可能

操作手順
1) 2mlチューブなどで菌体培養
2) cfg. 3500rpm, 5min, R.T.
3) 上清除去
4) NID-bufferを250μl入れる(培養スケールに合わせて適宜調整)
5) Voltexなどでよく混ぜる
6) 65℃, 5min放置
7) cfg. max, 10min, R.T.
8) ペレットを除去
9) NID-bufferと同量のisopropanolを加える(ここでDNAを沈殿させる)
10) Voltexなどでよく混ぜる
11) cfg. max, 15~20min, R.T.
12) 上清除去
13) 80% EtOHを300μl加える(不純物をエタノール溶液に溶かす, 遠心前にチューブをゆっくり回転させてチューブの内部全体をエタノールで洗う)
14) cfg, max, 5min, R.T.
15) 上清除去
16) 風乾
17) 1 x TE 50μlに溶かす