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: standard ratio of backbone to linker is ~1:100. This may need to be adjusted for non-standard conditions.)
• 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).
3. Transform 2.5 μL (or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.

Notes

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

1. List troubleshooting tips here.
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3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers, books, and websites

1. New England Biolabs

Contact

• Sean P Corum

or instead, discuss this protocol.