GigaPrep-DMAE-Column

1. Spin down cells from 5 liters of overnight culture.

2. Suspend bacteria in 125 ml of P1 (50 ml Tris/HCl, 10 MM EDTA, pH 8.0, 100 ÎŒg/ml RNase A)

3. Add 125 ml of buffer P2 (0.2 M NaOH, 1% SDS), mix. incubate at room temperature (RT) for 5 min.

4. Add 125 ml of buffer P3 (3 M potassium acetate, 2 M acetic acid), incubate on ice for 10 min.

5. Spin, use cheesecloth to remove junk from supernatant.

6. Add ER buffer (1 vol to 9 vol of supernatant), incubate 30 min at 4 oC.

7. Load solution to a DEAE silica gel column (26 mm×50 mm, 70-100 ÎŒm, 2.0 ÎŒmol/g) equilibrated with 75 ml of QBT buffer.

8. Wash column with 600 ml of QC buffer.

9. Elute DNA with 75 ml of QF buffer.

10. Add 52.5 ml of isopropanol and spin at 20,000×g for 30 min.

11. Dry the DNA pellet at RT in biosafety cabinet for 10 min.

12. Resuspend DNA in TE.

Endotoxin level: <300 I.U./mg of DNA

Endotoxin Removal ER Buffer
750 mM NaCl; 10% Triton® X 114 or Triton ® X 100; 40 mM MOPS, pH 7.0

QC Buffer
100 ml of 1 M Nacl, 15% ethanol, 50 mM MOPS, pH 7.0

QF Buffer
1.6 M NaCl , 15% ethanol, 50 mM MOPS, pH 7.0

Buffer P1
100 ÎŒg/ml RNase A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0

Buffer P2
200 mM NaOH, 1% SDS

Buffer P3
3.0M KAc, pH 5.5

Buffer QBT
750 mM NaCl, 50 mM MOPS, 15% isopropanol, pH 7.0, 0.15% Triton ® X 100

Buffer QC
1.0M NaCl, 50 mM MOPS, 15% isopropanol, pH 7.0

Buffer QN
1.6M NaCl, 50 mM MOPS, 15% isopropanol, pH 8.5

TE
10 mM Tris/HCl, 1mM EDTA, pH 8.0

STE
100 mM NaCl, 10 mM Tris/HCl, 1 mM EDTA, pH 8.0

diethylaminoethyl (DEAE)
diethylaminopropyl, dimethylaminoethyl (DMAE)

Ref:

Proc. Natl, Acad. Sci., USA, 76, p. 615-619