Paul.xu at 02:36, 5 June 2011

2011-06-05T02:36:26Z

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'''Low endotoxin plasmid Maxi-prep using cetyltrimethylammonium bromide and Tritonx-114'''

'''S I ''' 50 mM Tris-HCl, 10 mM EDTA, pH 8.0 

Optional: add 1 ml of 100 mg/ml thymolphthalein in ethanol to 100 ml S I solution 

'''S II''' 

0.2M NaOH and 1% SDS; 

'''S III ''' 

2.0M HAc, 0.6M KAc, 0.4M MnCl2 , 0.4 M CaCl2; 

 

'''Ca/PEG''' 

0.4 M calcium chloride, 40% PEG 8000, water. 

 

'''5% (w/v) CTAB''' Cetyl trimethylammonium bromide 

 

'''40% PEG 8000''' Polyethylene glycol 8000 

 

'''10% Triton X-114''' 

10g in 100 ml autoclaved water 

 

1. Grow 500 ml E. coli culture overnight at 37° C. with vigorous shaking;

2. Spin down the bacteria at <strike>4,500 g</strike> 6000 rpm for 5 min. Discard the supernatant;

3. Re-suspend the bacteria in 24 ml of S I, votex.

4. Lyse the bacteria by adding 24 ml of S II, mi '''gently.'''

5. Neutralize the lysate with 24 ml of S III, mix, incubate on ice for 5 min.

<strike>6. Optional: add 5% (w/v) CTAB to 0.18% final concentration, incubate 15 min at room temperature</strike>

6. Centrifuge the lysate at <strike>13,000 g</strike> 6000 rpm for 10 min. Transfer the clear supernatant to a new tube; Filter using cheesecloth to remove junk if you see white solid in supernatant.

7. Add 8 ml of 40% PEG 8000 (final conc is 4%) to the '''clarified lysate'''. Close the cap and mix the contents. Centrifuge the tube at 6,000 <strike>g</strike> rpm for 10 min. Discard the supernatant;

8. Dissolve the pellet in 0.9 ml of water and transfer to a 1.5 ml microfuge tube. Add 100 µl of Ca/PEG to the tube, wash tube with 0.9 ml water again to collect residual DNA, also add 100 ul of Ca/PEG. Close the tubes and invert several times to mix the content. Centrifuge the tube in a microfuge for 5 minutes to pellet the plasmid DNA. 

9. Add 0.5 ml of <strike>15% isopropanol</strike>  70% EtOH  to the tubes. Close the tubes and invert the tubes a few times;

10. Spin and pour off liquid.

11. Briefly spin the tubes in a microfuge to collect all liquid to the bottom. Remove all the liquid using a pipette;

12. Add 0.5 ml of water to each tube to dissolve the plasmid DNA, combine DNA solution;

13. Add 100 ul of 10% Triton X-114 to final concentration of 1% TritonX-114 to the plasmid DNA solution.

14. Incubate on ice for 10 min.

15. Incubate in a 42°C water bath or heating block for 10 min.

16. Spin at RT max speed for 5 min.

17. Collect supernatant in a 2.0 ml tube, add 0.7 vol (750 ul) of '''isopropanol''', incubate on ice for 10 min, spin to precipitate DNA.

18. Wash pellet with 0.35 ml of  '''70% EtOH''' and spin.

19. Air-dry pellet and dissolve DNA in TE.

20. Add 1 ul DNA to 50 ul water to measure DNA concentration, 

bacterial genomic DNA: < 10 ug/ mg endotoxin: < 50 EU/mg protein: < 10 ug/mg 

[[Category:Protocols]]

EndoToxin-Free Maxi Plasmid-Prep - Revision history

Paul.xu at 02:36, 5 June 2011