IGEM:Paris Bettencourt 2012/Protocols/Ligation

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This protocol is for ligating together two fragments of DNA, for example a vector backbone and an insert, prepared by restriction digest. The reagents listed below are from the Fermentas DNA Ligation Kit, but other kits that use the T4 ligase are similar. The best results are attained with a molar ratio of 1:3 (Vector:Insert). The spectrophotometer gives the DNA concentration in ng / ul. You can convert to moles / l by the following formula:

\frac{C [g/L]}{600*Length [bp]} = C [mol/L]

You can play with the ratio of vector to insert below, while keeping the total concentration vector + insert at 4 ul. The exact ratio is not critical as long as the insert is present in excess.

NOTE: The vector should be dephosphorylated and the insert must be phosphorylated. The means the insert should not have been treated with phosphatase.


Reagent
Volume
Vector
1 ul
Insert
3 ul
H20
4.5 ul
T4 Ligase Buffer (Fermentas)
1.0 ul
T4 Ligase Enzyme (Fermentas)
0.5 ul
Total volume:
10 ul


  • Flick the tube gently to mix.
  • Incubate at room temperature for 10 minutes.
  • The ligation reaction works best when transformed directly. It can be stored at 4 °C overnight, but not longer.
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