Used silica resin can be regenerated more than 50 times without affecting its capacity.
MATERIALS AND SOLUTIONS
Buffer-1 (Resuspension Buffer) (500 ml)
50 mM Tris-HCl (pH8.0) --------------------- 25 ml of 1 M Tris-HCl
10 mM EDTA -------------------------------- 10 ml of 0.5 M EDTA
10 mg RNase A (heat-treated) ---------------- 5 ml of RNase A (10 mg/ml)
Deionized H2O ------------------------------- 460 ml
Keep at 4oC.
Buffer-2 (Lysis Buffer) (500 ml)
200 mM NaOH ------------------------------- 20 ml of 5 M NaOH
1% SDS -------------------------------------- 50 ml of 10% SDS
Deionized H2O -------------------------------- 430 ml
Buffer-3 (Neutralization Buffer) (500 ml)
5 M Potassium acetate (pH4.8) ------------------ 245 g
Glacial acetic acid -------------------------------- 115 ml
Add deionized H2O to make a final volume of --- 500 ml
CRITICAL-Adjust pH to 4.8 with glacial acetic acid.
Equilibration Buffer (500 ml)
750 mM NaCl -------------------------- 75 ml of 5 N NaCl
50 mM MOPS (pH 7.0) ------------------ 50 ml of 0.5 M MOPS
15% Ethanol --------------------------- 75 ml of 100% Ethanol
0.15% Triton X-100 --------------------- 750 ul of 100% Triton X-100
DW ------------------------------------- 300 ml
Wash Buffer (500 ml)
1.0 M NaCl --------------------------- 100 ml of 5 M NaCl
50 mM MOPS (pH 7.0) ------------------- 50 ml of 0.5 M MOPS
15% Ethanol --------------------------- 75 ml of 100% Ethanol
DW ------------------------------------ 275 ml
Elution Buffer (500 ml)
1.25 M NaCl --------------------------- 125 ml of 5 M NaCl
50 mM Tris-HCl (pH 8.5) --------------- 25 ml of 1 M Tris-HCl
15% Ethanol --------------------------- 75 ml of 100% Ethanol
DW ------------------------------------ 275 ml
PROCEDURES
Column Regeneration
1. Rinse used silica-column with acetone. Fill up the column with acetone.
2. Rinse column with deionized water. Fill up the column with water.
3. Repeat acetone and water wash three times.
Minimum 3 repeat is essential to remove any cell debris.
Any remained cell debris in in the silica column decreases the plasmid yield.
4. Equilibrate column with 5-10 column volumes of Equilibration buffer.
Purification
5. Load the prepared sample from "step 13"
[ie., Isolation of Plasmid DNA from E. coli (Alkaline Lysis Method)].
6. Wash column with 10 column volume (use 20 ml for maxi column) of Wash buffer.
7. Elute plasmid DNA with 5 column volume (use 10 ml fo maxi column) of Elution buffer.