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EasyPrep Plasmid Miniprep Protocol

(Carry out all steps at room temperature)

Centrifugation Method

  1. Harvest 1-3 ml overnight bacterial culture by centrifugation at 5,000 g for 10 min (or 3,500 rpm for 20 min) in a tabletop centrifuge. Pour off the supernatant and blot the inverted tube on a paper towel to remove excess media.

  2. Add 250 µl Buffer A1 and completely resuspend bacterial pellet by vortexing or pipetting. Ensure that RNase A is added into Buffer A1 before use. Complete resuspension is critical for optimal yields.

  3. Add 250 µl Buffer A2, mix gently but thoroughly by inverting 5-6 times, incubate for approximately 2-4 min until the solution become slightly clear. Do not incubate longer than 5 min. Over-incubation causes genomic DNA contamination and plasmid damage. Buffer A2 may form precipitates below room temperature. Warm up at 37oC water-bath to dissolve the precipitates before use.

  4. Add 350 µl Buffer N1, mix immediately and thoroughly by inverting 5-6 times. Don’t vortex! It is critical to mix the solution well, if it appears conglobated, more mix is required.  

  5. Centrifuge at 13,000 rpm for 5 min at room temperature. During centrifugation, add 100 µl Column Buffer onto the DNA column, incubate for 2 min, centrifuge at 11,000 rpm for 30-60s. Note: a). If the lysate doesn’t appear clear, reverse the tube angle, centrifuge for 5 min and then transfer the lysate to the DNA  column. b). Column equilibration ensures the column having optimal binding capacity.

  6. Carefully transfer up to 750µl clear lysate into a DNA column with the collection tube (do not bring any floating material into the tube). Centrifuge at 11,000 rpm for 30-60s. Discard the flow-through. Note: when using end+ bacteria strains, such as JM series and HA201 derivatives, it’s important removes trace nuclease. Add 400 µl PB Buffer onto the DNA column, centrifuge at 11,000 rpm for 30-60 s. Discard the flow-through. Endstrains such as DH5a, Top10 and XL-1 blue do not require this step.

  7. Add 750 µl DNA Wash Buffer into the DNA column, centrifuge at 11,000 rpm for 30-60s. Discard the flow-through. Ensure that the 96-100% ethanol has been added to DNA Wash Buffer according to instructions before use.

  8. Centrifuge at 11,000 rpm for additional 1 min. It is critical to remove residual ethanol for optimal elution. Residual ethanol may interfere subsequent applications.

  9. Place the DNA column in a clean 1.5 ml microcentrifuge tube (Be careful not to transfer any of the ethanol with the DNA column). Add 50 µl Elution Buffer (TE buffer, pH8.5) to the center of the column, incubate for 1 min, centrifuge at 11,000 rpm for 1 min to elute the plasmid DNA. Elution buffer can be replaced with 10mM Tris.HCl, pH8.5 if EDTA interferes subsequent enzyme digestions. Proper pH is essential.

Vacuum Method

  1. Harvest 1-3 ml overnight bacterial culture by centrifugation at 5,000 g for 10 min (or 3,500 rpm for 20 min) in a tabletop centrifuge. Pour off the supernatant and blot the inverted tube on a paper towel to remove excess media.

  2. Add 250 µl Buffer A1 and completely resuspend bacterial pellet by vortexing or pipetting. Ensure that RNase A is added into Buffer A1 before use. Complete resuspension is critical for optimal yields.

  3. Add 250 µl Buffer A2, mix gently but thoroughly by inverting 5-6 times, the solution becomes slightly clear. Do not incubate longer than 5 min. Over-incubation causes genomic DNA contamination and plasmid damage. Buffer A2 may form precipitates below room temperature. Warm up at 37oC water-bath to dissolve the precipitates before use.

  4. Add 350 µl Buffer N1, mix immediately and thoroughly by inverting 5-6 times. Don’t vortex! It is critical to mix the solution well, if it appears conglobated, more mix is required.  

  5. Centrifuge at 13,000 rpm for 5 min at room temperature. During centrifugation.

  6. Prepare the vacuum manifold as instructed by manufacturer. Insert DNA columns into the luer connector of the vacuum manifold. Add 100 µl Column Buffer onto the DNA column, incubate for 2 min, switch on vacuum to draw the solution through the columns. This step ensures column having optimal binding capacity and is needed especially when the kit is stored for a while (over 6 months).

  7. Carefully transfer up to 750 µl clear lysates into Miniprep DNA columns (Do not bring any floating material into the tube). Switch on vacuum to draw the solution through the columns completely, and then switch off vacuum. Note: when using end+ bacteria strains, such as JM series and HA201 derivatives, it’s important to remove trace nuclease. Add 400 µl PB Buffer into the DNA column, centrifuge at 11,000 rpm for 30-60 s. Discard the flow-through. Endstrains such as DH5a, Top10 and XL-1 blue do not require this step.

  8. Add 750 µl DNA Wash Buffer into the DNA columns. Switch on vacuum source to draw the solution through the columns completely, and then switch off vacuum source.  Ensure that the 96-100% ethanol has been added to DNA Wash Buffer according to instructions before use.

  9. Transfer the DNA columns to 2 ml collection tubes. Centrifuge at 11,000 rpm for 1 min . It is critical to remove residual ethanol for optimal elution. Residual ethanol may interfere subsequent applications.

  10. Place each DNA column in a clean 1.5 ml microcentrifuge tube (Be careful not to transfer any of the ethanol with the DNA column). Add 50-100 µl Elution Buffer (TE buffer, pH8.5) to the center of the column, incubate for 1 min, centrifuge at 11,000 rpm for 1 min to elute the plasmid DNA. Elution buffer can be replaced with 10mM Tris.HCl, pH8.5 if EDTA interferes subsequent enzyme digestions.

DNA storage

Plasmid DNA is stable in TE Buffer if stored at -20°C or below .

 

Yield and quality of DNA

Determine the DNA absorbance of an appropriate dilution (20 to 50 fold) of the DNA at 260 nm and 280 nm. The DNA concentration is calculated as below:

DNA concentration = A260 x 50 x dilution factor (µg/ml)

 

 

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