Corum:Miniprep

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Procedure)
m (Notes)
Line 42: Line 42:
#Anecdotal observations that might be of use to others can also be posted here.   
#Anecdotal observations that might be of use to others can also be posted here.   
-
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
+
Please sign your name to your note by adding to the beginning of your tip.
==References==
==References==

Revision as of 15:41, 25 June 2012

  • SC 15:38, 25 June 2012 (EDT):

Contents

Overview

Miniprep plasmid DNA isolation using Sigmas's GeneElute Plasmid Miniprep Kit.

Materials

  • Miniculture
  • 1-5 mL overnight miniculture cells (E. coli)
  • GeneElute miniprep column
  • Resuspension buffer
  • Lysis solution
  • Stop solution
  • Column preparation solution
  • Wash solution
  • Optional wash solution
  • Elution buffer or H2O


Procedure

  1. Harvest the miniculture cells in a 2 ml tube by centrifugation. Remove all media by pipetting.
  2. Resuspend pellet in 200 μL resuspension buffer by pipetting and vortexing.
  3. Add 200 μL lysis solution. Gently invert 8 times. Incubate RT 4 mins.
  4. Add 350 μL stop solution. Gently invert 8 times. Wait a moment, and invert another 8 times.
  5. Spin 24,000g 3 min.
  6. In the meantime, prepare the column system. Add 500 μL column preparation solution and spin maximum speed 1 min in a tabletop centrifuge.
  7. Leaving the cellular debris behind, remove the supernatant by decanting or pipetting and apply to the column membrane. Spin 30 s maximum speed. Discard the flowthrough.
  8. Apply 500 μL optional wash solution to the column. Spin 30 s maximum speed. Discard the flowthrough.
  9. Apply 500 μL wash solution to the column. Spin 30 s maximum speed. Discard the flowthrough.
  10. Spin 1.5 min maximum speed to dry. Remove column and place in a new 1.5 ml tube.
  11. Apply 50-100 μL water or buffer EB to the column (make sure not to touch the membrane). Incubate RT 10 min.
  12. Elute DNA by spinning 2 min maximum speed.
  13. Optional: combine elutions of the same plasmid DNA.
  14. Optional: vacuum centrifuge at 60 °C to evaporate liquid down to desired volume (usually 50-100 μL).

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding to the beginning of your tip.

References

Relevant papers, books, and websites

  1. Sigma-Alrich

Contact

or instead, discuss this protocol.

Personal tools