Corum:T4 Ligation

• SC 15:16, 19 June 2012 (EDT):

Overview

Standard DNA ligation using NEB T4 ligase to form a vector.

Materials

For a 10 μL ligation reaction:

• 5 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
• 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 1:100. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
• 2 μL sterile ddH₂O
• 1 μL 10X ligation buffer
• 1 μL 10mg/ml BSA
• 0.5 μL T4 ligase

Procedure

1. Add the reaction components together in order listed above. Mix gently and spin.
2. Incubate 16 °C overnight (8 hr minimum).

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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References

Relevant papers, books, and websites

1. New England Biolabs

Contact

• Sean P Corum

or instead, discuss this protocol.