Editing 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 mmÃ50 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 20,000Ãg for 30 min.

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

12. Resuspend DNA in TE.

'''Endotoxin level''': &lt;300 I.U./mg of DNA

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

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

'''QF Buffer'''<br>1.6 M NaCl , 15% ethanol, 50 mM MOPS, pH 7.0

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

'''Buffer P2'''<br>200 mM NaOH, 1% SDS

'''Buffer P3'''<br>3.0M KAc, pH 5.5

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

'''Buffer QC'''<br>1.0M NaCl, 50 mM MOPS, 15% isopropanol, pH 7.0

'''Buffer QN'''<br>1.6M NaCl, 50 mM MOPS, 15% isopropanol, pH 8.5

'''TE'''<br>10 mM Tris/HCl, 1mM EDTA, pH 8.0

'''STE'''<br>100 mM NaCl, 10 mM Tris/HCl, 1 mM EDTA, pH 8.0

diethylaminoethyl (DEAE)<br>diethylaminopropyl, dimethylaminoethyl (DMAE)

'''Ref:'''

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



[[Category:Protocols]]