T4 Ligation-PDF

Overview

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

Materials

For a 10 μL ligation reaction:

• 3 μL sterile ddH₂O
• 1 μL 10X ligation buffer
• 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
• 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
• 0.5 μL T4 ligase

(Note: ratio of backbone to linker given here is ~1:100. This may need to be adjusted in some cases.)

Procedure

1. Add the reaction components together in order listed above. Mix gently and spin.
2. Include -linker control reaction (sub sterile ddH₂O for linker).
3. Incubate 16 °C overnight (8 hr minimum).
4. Transform 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.