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œœœœœ@Standard ligation@œœœœœ



DNA fragments from the same or different sources can be covalently joined (ligated) with T4 DNA ligase. The ligated DNA can then be transformed, transfected, or in vitro packaged. The optimal ratio of vector to insert DNA, and their absolute concentrations in the ligation reaction depends on the type of ligation; i.e. sticky-end or blunt-end, lambda, cosmid, plasmid or M13, phosphatased or not, isolated DNA or a mixed size population. In general, however, for cloning or subcloning restriction fragments, or several restriction fragments mixed together, the ratio (5 insert:1 vector) and concentrations (40-50ng vector DNA/20ul) given here works well.
Blunt-end ligations generally are much more difficult than sticky-end ligations, especially if the blunt-ends are generated by S1-nuclease (because S1 often leaves a single base overhang), and require more ligase, longer incubation time, and higher concentrations of DNA. Other conditions, such as spermidine concentration, can also affect the efficiency of blunt-end ligation. S1 treated DNAs should be end-repaired before ligating.
If the vector DNA has been treated with alkaline phosphatase, the vector:insert DNA ratio should be 1:1. This ratio can be obtained by increasing the amount of vector DNA used.
If the concentration of DNA is too high, there will be a tendency to insert 2 or more insert fragments into the vector molecule. This can be disasterous if a population of DNAs is being "banked". There will also be a tendency to form dimers, trimers, etc of the vector molecules, especially if they are not phosphatased. When screening your clones for inserts by electrophoresis of uncut plasmid or RF DNA, be on the lookout for clones that are exactly twice the size of the vector. If the concentration of DNA is too low, few of the colonies/plaques will have inserts; they will mostly be recircularized vectors.

  1. Materials

    1. 0.5M Tris, pH 7.8
      6.057 grams/100ml - pH with conc HCl

    2. 0.2M DTT
      0.6168 grams/10ml

    3. 1M MgCl2-7H2O
      20.33 grams/100ml

    4. 25mM spermidine-3HCl
      0.1591 grams/25ml

    5. 10mM ATP
      dissolve 0.0055 grams ATP in 1ml sterile ddH2O. Use a flamed spatula to weigh it out, to minimize DNase.

  2. Methods

    1. Make up 100ul 2X ligation buffer by mixing the following in a 500ul microfuge tube:
      20ul 0.5M Tris pH 7.8
      20ul 0.2M DTT
      20ul 10mM ATP
      2ul 1M MgCl2
      2ul 25 mM spermidine 3HCL
      0.25ul 20mg/ml BSA (nuclease-free)
      35.75ul ddH2O
      makes 100ul 2X

    2. Then mix up the following reaction mixture:
      10ul 2X ligation buffer
      Xul vector DNA (40-50ng)
      Yul insert DNA (3:1 molar ratio)
      Zul ddH2O
      19ul total volume

    3. Vortex, and add 1ul T4 DNA ligase. Incubate overnight at 15C.
    4. Transform or transfect competent host cells (prepared with CaCl2) by the method described in Maniatis, Molecular Cloning.

    5. FINAL LIGATION CONDITIONS
      50mM Tris, pH 7.8
      20mM DTT
      1mM ATP
      10mM MgCl2
      0.25mM spermidine 3HCl
      100ug/ml BSA
      2ug/ml vector DNA
      3:1 molar ratio insert DNA
      12 units ligase



BRL Product Profile: T4 DNA Ligase
New England Biolabs Data Sheet: T4 DNA Ligase
N. E. Biolabs "M13 Cloning/DNA Sequencing System" booklet 1982
NEN booklet "M13 Cloning Kit" 1983

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