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Latest revision as of 06:58, 19 October 2016

Restriction Enzyme Digest and Ligation · Benchling

Restriction Enzyme Digest and Ligation

Introduction

Notes - keep all reagents on ice. This protocol uses reagents from NEB. Protocol obtained from the iGEM Registry, modified to contain commercially available reagents. 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. Hazards: BSA: Highly flammable. Harmful if swallowed. Causes burns by all exposure routes. Reacts violently with water. Material is a low melting point solid that may become a liquid above room temperature. Target Organs: Respiratory system, gastrointestinal system, eyes, skin.

Materials

  • Reagents
    • dH2O (autoclaved)/ nuclease free H20 (which you don't need to autoclave)
    • NEB CutSmart Buffer
    • PstI
    • EcoRI
    • NEB Buffer 2.1
    • T4 DNA Ligase
    • T4 DNA Ligase Buffer
  • Equipment
    • 2016 Kit
    • Pipette + tips (p2, p20)
    • PCR tubes
    • Microcentrifuge tubes
    • Centrifuge
    • Thermal cycler
    • Ice and bucket/container

Procedure

  • Restriction Digest Protocol - Parts
  1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
  1. Add 2.5ul of NEBuffer 2.1
  1. Add 0.5ul of BSA.
  1. Add 0.5ul of EcoRI.
  1. Add 0.5ul of PstI.
  1. There should be a total volume of 20ul. Mix well and spin down briefly. Remember to balance your centrifuge.
  1. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid
  1. Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.
  • Restriction Digest Protocol - Backbone
  1. Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)
  • 5 ul NEB Buffer 2.1
  • 0.5 ul CutSmart Buffer
  • 0.5ul EcoRI - HF
  • 0.5ul PstI
  • 0.5ul DnpI
  • 18ul dH20
  1. Digest Plasmid Backbone
  • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
  • Add 4 ul of Enzyme Master Mix
  1. Digest 37C/30 min, heat kill 80C/20 min
  • Ligation
  1. Adjust your reaction amounts using the UT Dallas Ligation calculator: https://2011.igem.org/Team:UT_Dallas/ligation
  1. Add 2ul of digested plasmid backbone (25 ng)
  1. Add equimolar amount of digested insert (< 3 ul)
  1. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
  1. Add 0.5 ul T4 DNA ligase
  1. Add water to 10 ul
  1. Ligate 16C/30 min, heat kill 80C/20 min
  1. Transform with 1-2 ul of product
  • Leaving the Lab
  • As long as the gels are fully solidified they may go in the regular lab garbage bins.  Dispose of any excess buffer according to regulations  Disconnect power supply and ensure that all machinery is turned off
  • Reference
  • IGEM 2015
  • Changelog
  • 5/16/2016
  • 21/06/2016
  • 27/06/2016



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