Apr 27, 2020
LR Clonase
- Invitrogen1
- 1Thermofisher
Protocol Citation: Invitrogen 2020. LR Clonase. protocols.io https://dx.doi.org/10.17504/protocols.io.bfm9jk96
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2020
Last Modified: April 27, 2020
Protocol Integer ID: 36257
Add the following components to a 1.5-mL microcentrifuge tube at room temperature and mix: • 1–7 μL entry clone (50–150 ng) • 1 μL destination vector (150 ng/μL) • TE bufferǰ pH 8.0, to 8 μL
Thaw on ice the LR Clonase™ II enzyme mix for about 2 minutes. Vortex the LR Clonase™ II enzyme mix brefl¢ twice (2 seconds each time).
To each sample (step 1), add 2 μL of LR Clonase™ II enzyme mix to the reaction and mix well by vortexing brefl¢ twice. Microcentrifuge brefl¢ǯ
Return LR Clonase™ II enzyme mix to –20°C or –80°C storage.
Incubate reactions at 25°C for 1 hour. (overnight)
Add 1 μL of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly Incubate samples at 37°C for 10 minutes. Transform
Transform 1 μL of each LR reaction into 50 μL of One Shot™ OmniMAX™ 2 T1 Phage-Resistant Cells (Cat. no. C8540-03) (5 alpha e. coli). Incubate on ice for 30 minutes.
Follow electroboration protocol.
T1 Phage-Resistant Cells as described above. Plate 20 μL and 100 μL on LB plates containing 100 μg/mL ampicillin. Expected results An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated.