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Latest revision as of 06:58, 19 October 2016

Quantum Prep Plasmid Miniprep · Benchling

Quantum Prep Plasmid Miniprep

Introduction

Get started by giving your protocol a name and editing this introduction. Safety Precausions SDS (Safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets. PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.

Materials

  • Reagents
    • E. coli culture
    • Cell Resuspension Solution
    • Cell Lysis solution
    • Neutralization Solution
    • Quantum Prep Matrix
    • Wash buffer
    • Spin Filters
    • Wash tubes
    • Collection tubes
    • 9H2O or TE Buffer
  • Equipment
    • Centrifuge
    • Vortexer
    • Micropipetter

Procedure

  • How To
  1. Trasfer 1-2 mL of the E. coli with RFP to a micro test tube; centrifuge for 30 seconds at room temperature at 12 000 - 14 000 rmp. Remove the supernatant.
00:00:30
  1. Add 200 µL cell resuspension solution; vortex until cell pellet is completely resuspended.
  1. Add 250 µL cell lysis solution; mix by inverting 10 times (do not vortex)
  1. Split the solution into two spin columns, each 275 µL.
  1. Add 125 µL neutralization solution into each of the two columns. Mix by inverting 10 times (a visible percipitate should form), then centrifuge for 5 minutes at 12 000- 14000 rmp.
00:05:00
  • Acompact white debris pellet will form along the side or at the bottom of the tube. The supernatant (clear lysate) will contain the plasmid DNA.
  1. Insert each spin column into seperate 2 mL wash tube; shake and invert Quantum Prep matrix until completely suspended.
  1. Transfer supernant into spin column. Add 200µL Quantum Prep matrix ( 100µL into each spin collumn) and mix by pipetting up and down. Centrifuge for 30 seconds at 12 000 -14 000 rpm to pull fluid through the column.
00:00:30
  1. Remove spin column, discard fitrate and replace column. Add 500 µL wash buffer (250 µL into each spin column) and centrifuge for 30 seconds.
  1. Remove spin column, discard fitrate and replace column. Add 500 µL wash buffer (250 µL into each spin column) and centrifuge for 2 full minutes to remove all wash buffer.
00:02:00
  1. Remove spin column to a clean 1.5 mL tube. Add 100 µL H2O or TE buffer (50 µL each) and centrifuge at 12 000- 14 000 rpms for 1 minute.
00:01:00
  1. Discard the spin filter, label "IGEM- MiniPrepped plasmid-date- and store the eluted DNA at -20 oC.
  • Reference
  • IGEM 2015
  • Bio Rad, Quantum Prep Plasmid Miniprep Kit Instruction Manual; Catalog 732-6100
  • Changelong
  • 5/16/2016



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