Fast DNA ligation
You want to ligate two DNA strands. Classically, this is necessary to insert a gene in a plasmid during cloning.
- Mix the following components:
- 100 ng purified, linearised vector
- purified linear insert in 3 – 5 fold molar excess over vector
- fill up to 9 µl with double distilled water
- 10 µl 2 x fast ligation buffer
- 0.5 µl T4 DNA ligase (400 U/µl)
- Incubate for 10 min at room temperature and place on ice for transformation
- 2 x fast ligation buffer: 132 mM Tris pH 7.6, 200 mM MgCl2, 15% PEG (3350 – 8000), 2 mM DTT, 2 mM ATP; prepare aliquots and store at –20°C
This fast protocol is a replacement for any fast DNA ligation kit out there. You simply take a plain normal T4 DNA ligase and combine it with the PEG-containing buffer which speeds up the reaction.
Since DTT and ATP are not very stable, it is advisable to prepare single-use aliquots and rather discard the 2 x ligation buffer than freezing again.