Plasmid miniprep - Qiagen

Reagents

The DNA prepared can be used for sequencing, enzymatic digestion, subcloning, etc.

1. Day 1, seed bacteria to 3 ml LB liquid medium and culture overnight at 37C, RPM 220.

2. Day 2, transfer 1.5 ml bacteria to tubes and spin down at 5K RPM for 5 min at RT.

3. Remove supernatant and resuspend the pellet thoroughly with 250 ul P1 solution.

4. Add 250 ul buffer P2 and invert the tube 2-3 times gently. The solution becomes clear.

5. Add 350 ul buffer N3 and invert the tube 2-3 times gently. Spin down at 14000 RPM for 10 min at RT.

6. Carefully transfer the supernatant to a column. Spin down at 14K RPM for 1 min at RT. Discard flowthrough.

7. Apply 500 ul buffer PB and spin down at 14K RPM for 1 min at RT. Discard flowthrough.

8. Apply 700 ul buffer PE (with ethanol added). Spin down at 14K RPM for 1 min at RT. Discard flowthrough.

9. Spin down one more time at 14K RPM for 1 min at RT.

10. Transfer the column to a clean tube. Apply 30-50 ul TE buffer to the center of the column.Incubate for 1 min at RT.

11. Spin down to collect DNA at 14K RPM for 1 min at RT.


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Posted by ebiomethods Tue, 23 Dec 2008 10:43:12
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