Plasmid MiniPrep NID

NID miniprep plasmid isolation procedure

1. Spin down 2 ~ 5 ml of  bacterial cultures.

2. Add 200 ul extraction buffer, transfer to 1.5 ml microcentrifuge tube. Resuspend into homogenous solution.

3. Incubate at 65°C for 5 min.

4. Spin at 13000 rpm for 5 ~ 10 min.

5. Remove pellet using pippet tip.

6. Add 150 µl isopropanol, mix.

7. Spin at 13000 rpm for 10 min at RT.

8. Discard liquid.

DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions

9. Add 100 70% ethanol. Gently remove ethanol.

10. Dissolve DNA pellet in 50 µl TE buffer.

DNA can be used for restriction digestion. For sequencing: DNA may have to be purified using silica spin column.

Extraction Buffer

5% sucrose

50 mM EDTA

50 mM Tris pH 8

0.75 M NH₄Cl or Ammonium acetat (NH4Ac)

20 mM CaCl₂

0.5% IGEPAL CA-630 (Nonidet-P40) or Triton X-100

Add lysozyme 100 µg/ml, and RNase A 25 µg/ml from 100X enzyme stock (10 mg/ml lysozyme and 2.5 mg/ml of RNase A  in 50% glycerol, 50 mM Tris pH 8).

2013/1/9

digest with Nde1+Bgl11