Team:Ionis Paris/Protocol 4

Protocol 4 : Digestion and Ligation

Aim: Perform double strand cuts in a DNA sequence and ligate two DNA sequences together

Digestion

In a 1.5mL Eppendorf tube, add:

  • 100ng DNA

  • 1µL Enzyme 1

  • 1µL Enzyme 2

  • 2µL 10X appropriate buffer

  • Qsp 20µL ultrapure water

    • In a control tube, add the same components, but replace DNA with ultrapure water

    Mix gently and incubate under agitation at 37°C for 1h

    NB : Buffer to use

    Enzyme 1 Enzyme 2 Buffer
    SpeI PstI Cutsmart
    XbaI PstI NEBuffer 3.1
    EcoRI PstI NEBuffer 3.1

    Purification

    Gel purification : Run samples on electrophoresis gel and purify appropriate insert and vector.
    See the appropriate protocol (Protocol 3 : Electrophoresis )
    PCR purification : See the appropriate protocol (Protocol 8 : PCR and PCR purification)

    Ligation

    Add the following reaction in a microcentrifuge tube, respect the order (T4 DNA Ligase should be added last)
    Molar ratios were calculated using NEB BioCalculator (http://nebiocalculator.neb.com/#!/ligation).
    Molar ratio of 1:3 vector to insert shown

    Components 20µL reaction
    Nuclease free water to 20µL
    10X T4 DNA ligase buffer 2µL
    Vector DNA 50ng (0.020 pmol)
    Insert DNA Xng (0.060 pmol)
    T4 DNA Ligase 1µL

    Gently mix the reaction by pipetting up and down and microcentrifuge briefly.
    Incubate at 16°C overnight or room temperature for 1 hour
    Chill on ice and transform 5μl of the ligation product into 50μl competent cells.