Quick Ligation Protocol (M2200)
Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.
(Quick Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)
Use NEBiocalculator to calculate molar ratios.
|COMPONENT|| 20 μl REACTION
| Quick Ligase Reaction Buffer (2X)*
|| 10 µl
| Vector DNA (4 kb)
|| 50 ng (0.020 pmol)
| Insert DNA (1 kb)
|| 37.5 ng (0.060 pmol)
| Nuclease-free Water
|| to 20 µl
| Quick Ligase
|| 1 µl
*The Quick Ligase Reaction Buffer should be thawed and resuspended
at room temperature.
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. Incubate at room temperature (25°C) for 5 minutes.
4. Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells. Alternatively, Store at -20°C.
5. Do not heat inactivate – heat inactivation dramatically reduces transformation efficiency.
This tutorial describes the use of the NEBioCalculator web tool to optimize the molar ratio between vector and insert DNA for use in a ligation reaction.