www.bioland-sci.com

EasyPrep Plasmid Midiprep Protocol

Carry out all steps at room temperature
  • Centrifugation method
  1. Harvest 50-60 ml overnight cultured bacterial by centrifugation at 3,500rpm for 10 min at room temperature. Pour off the supernatant and inverte tube on paper towels to remove residual medium.
  2. Add 2.5 ml Buffer A1 and completely resuspend bacterial pellet by vortexing or pipetting. Ensure that RNase A has been added into Buffer A1 before use. Complete resuspension is critical for optimal yield.
  3. Add 2.5 ml Buffer A2, mix gently but thoroughly by inverting 5-6 times with slightly shaking. Incubate for 3-4 min to obtain a slightly clear lysate. 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.
  4. Add 1 ml Buffer A3, mix immediately but thoroughly by inverting 5-6 times. Don’t vortex. It is important to mix the solution well, if the mixture still appears conglobated, more mix is required.
  5. Use one of the two methods below to clear the lysates:
  • High speed centrifuge: Transfer the lysate to a high speed centrifuge tube and centrifuge at 13,000 rpm (14,000-18,000 g) for 15 min at room temperature! Transfer the cleared lysate to a 50 ml conical tube.
  • EasyFilter syringe: place the EasyFilter syringe  to a  clean  50  ml conical tube with the plunger removed. Pour the lysate into the filter syringe barrel. Allow the lysate to sit for 5 min. The white precipitates should float to the top. Hold the filter syringe barrel over the 15 ml tube and gently insert the plunger to expel the cleared lysate to the tube. Stop when feel resistance. some of the lysate may remain in the flocculent precipitate.
  1. Add 2 ml Buffer A3 and 2.5 ml Ethanol (96-100%). Mix well by inverting 5-6 times.
  2. Column preparation: add 1 ml Column Buffer to the column, incubate for 2-3 min, centrifuge at 3,500 rpm for 2-3 min. The column equilibration ensures the column having optimal binding capacity. Especially when the kit is stored over 6 month or the yield declines noticeably. 
  3. Transfer up to 4.5 ml lysate/ethanol mixture into a DNA column with the collection tube. Centrifuge at 3,500 rpm for 3-5 min. Discard the flow-through.
  4. Add 4.5 ml 70% ethanol into the column, centrifuge at 3,500 rpm for 3-5 min. Discard the flow-through.
  5. Repeat Step 9.
  6. Centrifuge at 3,500 rpm for 5 min. This step removes residual ethanol which is critical for DNA elution. Residual ethanol may also interfere subsequent applications.
  7. Place the column in a sterile 50 ml tube. Add 0.25-0.5 ml Elution Buffer (TE buffer, pH8.5) to the center of the column. Incubate for 1 min.  Elute the DNA by centrifugation at 3,500 rpm for 2-5 min. If higher DNA concentration is needed, use less elution buffer (i.e. 0.25 ml). Note: EDTA in elution buffer is important for DNA stability, especially when plasmid DNA is used for transfection.
  8. Add the eluate from last step back to the column, elute one more time. This will increase the yield. Alternatively, elute with another 0.25-0.5 ml Elution Buffer into a new 15 ml tube. Two elutions give rise to maximum DNA yield. Store DNA at –20oC.
Vacuum method
  1. Harvest 50-60 ml 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.
  2. Add 2.5 ml Buffer A1 and completely resuspend bacterial pellet by vortexing or pipetting. Ensure that RNase A has been added into Buffer A1 before use. Complete resuspension is critical for optimal yield.
  3. Add 2.5 ml Buffer A2, mix gently but thoroughly by inverting 5-6 times with slightly shaking. Incubate for 3-4 min to obtain a slightly clear lysate. Complete lysis is critical for DNA yield. The mixture of completely lysed bacteria looks transparent. Do not incubate longer than 5 min. Over-incubating causes genomic DNA contamination and plasmid damage.
  4. Add 1 ml Buffer A3, mix immediately but thoroughly by inverting 5-6 times. It is important to mix the solution well, if the mixture still appears conglobated, more mix is required.
  5. Use one of the two methods below to clear the lysates:
  • High speed centrifuge: Transfer the lysate to a high speed centrifuge tube and centrifuge at 13,000 rpm (14,000-18,000 g) for 15 min at room temperature! Transfer the cleared lysate to a  50 ml conical tube.
  • EasyFilter syringe: place the EasyFilter  syringe  to a  clean  50  ml conical tube with the plunger removed. Pour the lysate into the filter syringe barrel. Allow the lysate to sit for 5-10 min. The white precipitates should float to the top. Hold the filter syringe barrel over the 50 ml tube and gently insert the plunger to expel the cleared lysate to the tube. Stop when feel resistance. some of the lysate may remain in the flocculent precipitate.
  1. Add 2 ml Buffer A3 and 2.5 ml Ethanol (96-100%). Mix well by inverting 5-6 times. 
  2. Column preparation: add 1 ml Column Buffer to the column attached to the vacuum manifold incubate for 2-5 min, switch on vacuum source to draw the solution through the columns completely. This step ensures the column having optimal binding capacity. Especially when the kit is stored over 6 month or the yield declines noticeably.
  3. Transfer up to 4.5 ml lysate/ethanol mixture to the column, switch on vacuum to draw the solution through the columns completely, and then switch off. Apply the remaining lysate/ethanol mixture.
  4. Add 4.5 ml 70% ethanol into the column. Switch on vacuum source to draw the solution through the columns completely, and then switch off vacuum source.
  5. Repeat Step 9, leave the vacuum on for 2 min and then switch off. Removal of residual ethanol is critical for DNA elution. Residual ethanol may interfere subsequent applications. Increase the time 5-10 min if ≥10 samples are processed at same time.
  6. Detach the column from the manifold, wipe off any trace ethanol and put the column to a clean 50 ml conical tube.
  7. Add 0.25-0.5 ml Elution Buffer (TE buffer, pH8.5) to the center of the column, incubate for 1 min, elute the DNA by centrifugation at 3,500 rpm for 2-5 min. If higher DNA concentration is needed, use less elution buffer (i.e. 0.25 ml).
  8. Add the eluate from last step back to the column, elute one more time. This will increase the yield. Alternatively, elute with another 0.25-0.5 ml Elution Buffer into a new 15 ml tube. Two elutions give rise to maximum DNA yield. Store DNA at –20oC
The DNA is ready for down stream application such as cloning or transfection. If higher DNA quality and concentration is desired, such as for primary cultured cell or some cell line transfection, proceed to the next step. It’s also highly recommended to remove the endotoxin if the DNA is used for primary cultured cell transfection or microinjection.
 
(Updated 02-2017)
 
Copyright © 2021 Bioland Scientific, LLC. All rights reserved. Powered by Zen Cart. Hosted by Siteground.
FedEx service marks used by permission.