Plasmid MaxiPrep by Q Sepharose

Most economical, but need to optimize to remove endotoxin.

Column: 20 ml of Q-Sepharose for 300 ml bacterial culture

Wash Buffer:

1. 40% isopropanol and 1.5 M NaCl

2. 30% isopropanol  1M NaCl

3. 45% isopropanol 0.75M

Qiagen Q column:

RNA will be eluted from column at 1.0 M NaCl at pH7. DNA will be eluted at 1.4 ~ 1.6 M NaCl. pH is very critical.

Buffer QC

1.0 M NaCl
50 mM MOPS
Adjust pH to 7.0 with HCl
15% (v/v) isopropanol
Store indefinitely at room temperature

Buffer QF

1.25 M NaCl
50 mM Tris⋅Cl
15% isopropanol
Adjust pH to 8.5 with HCl
Store indefinitely at room temperature

Buffer QBT

750 mM NaCl
50 mM 3-(N-morpholino)propanesulfonic acid (MOPS)
Adjust pH to 7.0 with HCl
15% (v/v) isopropanol
0.15% (v/v) Triton X-100
Store indefinitely at room temperature

Endotoxin Removal

Dilute Eluted DNA 1:1 with 4M ammonium sulfate buffer then loaded onto a 2.6×2.5 cm Octyl HIC column equilibrated in 2M ammonium sulfate buffer.