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EndoFree Plasmid midiprep-II protocol

  1. Inoculate 100-125 ml LB containing appropriate antibiotic with 50 µL fresh starter culture. Grow at 37oC for 14-16 h with vigorous shaking. Prepare a starter culture by inoculating a single colony from a freshly grown selective plate into 1-2 ml LB medium containing the appropriate antibiotic. Grow at 37°C for 6-8 h with vigorous shaking (~300 rpm).

  2. Harvest the bacterial by centrifugation at 5,000 g for 10 min at room temperature. Pour off the supernatant and blot the inverted tube on paper towels to remove residual medium completely.  

  3. Add 5 mL Buffer A1 to bacterial pellet and completely resuspend by vortexing or pipetting. Note: Complete resuspension is critical for optimal yield. 

  4. Add 5 mL Buffer B1, mix thoroughly by inverting 6-10 times. Incubate at room temperature for 2-4 min to obtain a slightly clear lysate. Buffer B1 may form precipitation below room temperature. If solution becomes cloudy, warm up at 37-50oC to dissolve before use. Complete lysis is critical for DNA yield. The mixture of completely lysed bacteria looks transparent. Do not incubate longer than 5 min. Over-incubation causes genomic DNA contamination and plasmid damage. 

  5. Add 1.5 mL Buffer N3, mix immediately and  thoroughly by inverting 6-10 times. It is critical to mix the solution well.   Note: if the mixture still appears conglobated, brownish or viscous, more mixing is required to completely neutralize the solution. 

  6. Two options for clearing the lysates:

    1. High speed centrifuge: Transfer the lysate to a high speed centrifuge tube and centrifuge at 10,000 rpm for 20 min at room temperature! Transfer the cleared lysate to a 50 ml conical tube.

    2. ezFilter syringe:  Place the ezFilter syringe  to a  centrifuge tube in a tube rack. Using a serological pipet to transfer most of the lysate, avoid only a small portion of the major precipitates, to the barrier of the syringe.  Allow the lysate to incubate for 5 min. Gently insert the plunger to expel the clear lysate.  

  7. Add 5 mL Buffer RET and mix well.  

  8. Transfer the sample to a DNA midiprep-II column and centrifuge at 3,500 rpm for 3-5 min. Discard the flow-through.  

  9. Add 10 mL DNA Wash Buffer to the column and centrifuge at 3,500 rpm for 3-5 min. Discard the flow-through. Repeat once. Make sure ethanol is added before use.

  10. Centrifuge the column at 3,500 rpm for 10 minutes to remove residual ethanol. Important: Removal of residual ethanol is critical for DNA elution. If swing bucket centrifuge can’t reach the speed, use the fixed-angle high speed centrifuge. 

  11. Transfer the column to a clean 50 mL conical tube and add 0.5-1 ml EndoFree Elution Buffer (H2O) to the center of the column and incubate for 1 minute at room temperature. Elute the DNA by centrifugation at 3,500 rpm for 5 min. Use less elution buffer if higher DNA concentration is desired. 

  12. Add the eluted DNA back to the column and centrifuge at 3,500 rpm for 5 min. The first elution normally yields approximately 70% of the DNA. The second elution yields another 20% of the DNA bound to the column.

 The DNA is ready for downstream applications such as transfection of endotoxin sensitive cell lines, primary cell lines and microinjections.

 
(Updated 05/2013)
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