Difference between revisions of "Team:Toronto/Experiment-Monarch Miniprep Protocol"

 
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Latest revision as of 06:58, 19 October 2016

Monarch Miniprep Protocol · Benchling

Monarch Miniprep Protocol

Introduction

NEB #T1010 ( https://www.neb.com/products/t1010-monarch-plasmid-miniprep-kit ) Monarch protocol for miniprep as described by the protocol card found in its kit

Materials

  • Sample
    • 95% ethanol
      • Nuclease Free H20 or MilliQ Water
        • Monoarch Miniprep Kit
          • Plasmid Resuspension Buffer (B1)
          • Plasmid Lysis Buffer (B2)
          • Plasmid Neutralization Buffer (B3)
          • Plasmid Wash Buffer 1
          • Plasmid Wash Buffer 2
          • DNA Elution Buffer

        Procedure

        • Before you begin
        • - All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM)
        • - Add 4 volumes of ethanol (>95%) to one volume of Plasmid Wash Buffer 2
        • CRITICAL
          Only do this if you are using the Monarch miniprep kit for the first time
        • - If precipitate has formed in Lysis Buffer (B2), incubate at 30-37oC, inverting periodically to dissolve
        • - Store Plasmid Neutralization Buffer (B3) at 4oC after opening (store on ice or in fridge)
        • Protocol
        1. Pellet 1-5ml bacterial culture by centrifugation for 30 seconds. Discard supernatent
        • - Recommend 1.5ml in a microcentrifuge tube
        • - If cells grown in 6mL liquid cultures overnight, do 1.5mL twice (discardign supernatent after first centrifuge and adding another 1.5mL into the same tube) and begin pre-warming nuclease free water (nf-H2O) at 37oC
        1. Resuspend pellet in 200ul Plasmid Resuspension Buffer (B1)
        • - Vortex or pipet to ensure cells re completely resuspended.
        • - There should be no visible clumps
        1. Add 200ul Plasmid Lysis Buffer (B2), gently invert tube 5-6 times and incubate at room temperature for 1 minute
        • - Colour should change to dark pink and solution will become transparent and viscous
        • CRITICAL
          DO NOT VORTEX
        1. Add 400ul of Plasmid Neutralization Buffer (B3), gently invert tube until neutralized and incubate at room temperature for 2 minutes
        • - Sample is neutralized when colour is uniformly yellow and precipitate forms
        • CRITICAL
          DO NOT VORTEX
        1. Centrifuge lysate for 2-5 minutes
        1. Carefully transfer supernatent to spin column and centrifuge for 1 minute and discard the flow-through
        1. Re-insert column in the collection tube and add 200ul of Plasmid Wash Buffer 1 then centrifuge for 1 minute
        • - Discarding flow-through is option
        1. Add 400ul of Plasmid Wash Buffer 2 and centrifuge for 1 minute
        • - Recommend do a dry spin for 1 minute after this step but not absolutely necessary
        1. Transfer column to a clean 1.5ml microfuge tube
        • - Use care to ensure that the tip of the column does not come into contact with the flow-through
        • - If any doubt, do a dry-spin
        1. Add 30ul or more (recommend 50uL) of DNA Elution Buffer to center of matrix then wait for 1 minute and spin for 1 minute to elute DNA
        • - Nuclease-free water (pH 7-8.5) can be used instead of DNA Elution Buffer
        • - For larger size DNA (10kb or more), heating elution buffer to 50oC prior to use



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