LIC PROTOCOLS

LIC* Generator

*Ligation Independent Cloning

What is LIC ?

  • PCR
    • First of all, generate your primers using the LIC generator
    You can make simple LIC or introduce tags, restriction site,
    protease cleavage sites at the N-terminus or C-terminus of your insert.

    For PCR, use PHUSION DNA polymerase (Finnzyme ).
    This engineered polymerase is more robust than Pfu Ultra DNA polymerase
    for making the vector part in reasonable amounts

    Remarks

    We did not succeed making the vector with Pfu Ultra    .

    Cycling program PhusionV
    (vector)
    98°C 30s

    98°C 10s
    60°C 10s
    72°C 3 min (30s/kb)

    35 cycles

    72°C 10 min
    15°C hold

    PCR mix

    Mix1(primers)
    		Mix2(enzyme)
    Water6032
    5xPhusion Buffer40
    2.5 mM dNTP16
    Primer 120
    Primer 220
    Template DNA4
    DMSO6
    Phusion Polymerase2
    Final volume100100

    Remarks

    There is no need to linearize the vector
    A volume of 200 microl PCR mix is sufficient for subsequent purification steps

  • Gel purification of PCR product

    • Use Qiagen like purification kit after precipitation of the DNA and loading on a 1% agarose gel
    Elution with Elution buffer (generally Tris pH 8.0 or 8.5 is compatible with subsequent steps)

  • Digestion of parental DNA with Dpn1 and Action of T4 DNA polymerase


    • We used NEB buffer 2 in which Dpn1 AND T4 polymerase work for 20 min at 37%C in the presence of the correct deoxynucleotide.

    Protocol

    ReagentVolume (microl)
    Purified PCR product10
    T4 DNA polymerase Buffer10
    T4 DNA polymerase (1U/microl)1
    DpnI 1
    dGTP or dCTP 1OO mM1
    BSAx100 (with restriction enzymes)1
    H20qsp 100


  • Inactivation of enzymes and spin purification
    • Heating 10 min at 65°C
    Purification on spin column (Qiagen or equivalent)
  • LIC step

    • Remark

    We used the T4 DNA ligase Buffer that resembled the published buffer
    ATP is required to stabilize the duplexes (but some people use EDTA instead) !

    Definition of LIC buffer

    5xT4 DNA ligase buffer~10xLIC buffer
    250 mM Tris-HCl pH 7.625 mM
    50 mM MgCl25 mM
    5 mM ATP0.5 mM
    5 mM DTT0.5 mM(not required)
    25% PEG-80002.5%(not required)


    Protocol

    Add beta-mercaptoethanol 4 mM (dilution 1/320) to LIC buffer
    Mix : 9 ng/vector + 24 ng/insert or H20 (as negative control)
    in 20 microl containing 2 microl of LIC buffer.
    Incubation 1h at room temperature.
  • Transformation
    • 5 microl of each mix are used to transform TOP10 competent cells
    (or any competent cells) with classical protocol.
  • Analysis of transformant clones
    • Compare the number of colonies with those of the control reaction,
    miniprep and sequence 2 or more clones depending on the background.

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