Editing Plasmid MaxiPrep Silica Oxide (section)

== Protocol 2  ==

'''P1''': 50 mM Tris-HCl, 10 mM EDTA, pH 8.0, 50 ug/ml RNaseA (Sigma R4642), Store at 4 C<br>'''P2''': 0.2M NaOH, 1% SDS (4 g NaOH, 5 g SDS and distilled water to 500 ml)<br>'''N3''': 4.2 M guanidine hydrochloride and 0.9 M potassium acetate, pH 4.2 

or&nbsp;1 M Guanidine Hydrochloride<br>'''WS''': 100 mM NaCl, 10 mM Tris-HCl, 80% ethanol, pH 7.5<br>'''EB''': 10mM Tris-HCl pH 8.5, autoclave and remeasure the pH 

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1. Harvest 5 ml bacterial cells by centrifuging 1-2 min, 10,000-13,000 rpm. <br>2. Discard supernatant (aspirate or pour off) and resuspend pellet in 250 uL P1 by vortexing.<br>3. Add 250uL P2 and invert 5 times to mix (wait at least 2 min but no longer than 5 min). Do NOT vortex.<br>4. Add 350uL N3 and invert 5 times to mix. 

'''If P1 without RNAse is used, add 10 ul of an RNaseA solution (1.7 mg/mL) and incubate for 5 min at<br>room temperature.'''<br>5. Put on cold metal block (-20oC) for 5 min, or dry ice for 1 min. 

6. Spin 13,000 rpm for 5 min to pellet. Transfer the supernatant to a fresh tube. 

'''If endotoxin should be removed (or reduced):''' 

Add 100 uL buffer '''F4 (2.5% SDS in isopropanol)''' to precipitate LPS. Mix by votex, incubate at 4°C for 5 min then -20°C for 15 min. Spin for 10 min at maxispeed (microcentrifuge)<br><br>7. Add 25 µl silica matrix, mix well and let the tube sit at room temperature for 2 min.<br>8. Pellet the matrix by centrifugation for 20 sec at 13,000 rpm. Pour off the supernatant and gently tap the <br>inverted tube against a paper towel to drain the liquid.<br>9. Wash the matrix by resuspending with 750 µl WS and vigorously vortexing.<br>10. Repeat step 7 and 8 to wash glass matrix again.<br>11. Pellet the matrix by centrifugation for for 20 sec at 13,000 rpm. and remove the supernatant by pipetting.<br>12. Slowly blow air to dry the matrix (it takes less than 1 min), or you can leave the tube on bench for 10-15<br>minutes.<br>13. Add 55 µl EB to resuspend the pellet by brief vortexing and place the microfuge tube at 80°C for 2 min.<br>14. Centrifuge at 13,000 rpm for 2 min and transfer ~50 µl supernatant containing the eluted plasmid DNA to another tube. Apply DNA to downstream reaction or store at -20°C. 

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