Editing Plasmid MiniPrep NID

NID miniprep plasmid isolation procedure 

1. Spin down 2 ~ 5 ml of&nbsp; bacterial cultures.<br> 

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

3. Incubate at 65°C for 5 min.<br> 

4. Spin at 13000 rpm for 5 ~ 10 min.<br> 

5. Remove pellet using pippet tip.<br> 

6. Add 150 µl isopropanol, mix.<br> 

7. Spin at 13000 rpm for 10 min at RT.<br> 

8. Discard liquid.<br> 

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

9. Add 100 70% ethanol. Gently remove ethanol.<br> 

10. Dissolve DNA pellet in 50 µl TE buffer. 

<br> 

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

<br> '''Extraction Buffer'''<br> 

5% sucrose<br> 

50 mM EDTA<br> 

50 mM Tris pH 8<br> 

0.75 M NH<sub>4</sub>Cl or Ammonium acetat (NH4Ac)<br> 

20 mM CaCl<sub>2</sub><br> 

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

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&nbsp; in 50% glycerol, 50 mM Tris pH 8).<br> 

2013/1/9

digest with Nde1+Bgl11

[[Image:V19_digest_1-9-2013.JPG]]

[[Category:Protocols]]