Oct 29, 2020
LR Clonase Reaction for Multisite Gateway Cloning
- 1Brigham Young University
Protocol Citation: Ken Christensen 2020. LR Clonase Reaction for Multisite Gateway Cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.bn8gmhtw
Manuscript citation:
https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0001087_GatewayLR_ClonaseIIPlusEnzymeMix_UG.pdf&title=VXNlciBHdWlkZTogR2F0ZXdheSBMUiBDbG9uYXNlIElJIFBsdXMgRW56eW1lIE1peA==
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: In development
We are still developing and optimizing this protocol
Created: October 29, 2020
Last Modified: October 29, 2020
Protocol Integer ID: 44008
Abstract
Gateway® LR Clonase™ II Plus
Gateway® LR Clonase™ II Plus enzyme mix is a proprietary enzyme formulation specifically designed for MultiSite Gateway® and MultiSite Gateway® Pro. Gateway® LR Clonase™ II Plus enzyme mix contains the bacteriophage lambda recombination proteins Integrase (Int) and Excisionase (Xis), and the E. coli encoded protein Integration Host Factor IHF) (1), and reaction buffer provided in a single mix for convenient reaction set up. Gateway® LR Clonase™ II Plus enzyme mix promotes in vitro recombination between attL- and attR-flanked regions on entry clones and destination vectors to generate attB-containing expression clones.
Guidelines
General Recommendations and Guidelines
- We recommend using plasmid DNA purified with the PureLink™ HiPure Plasmid Midiprep Kit (Catalog no. K2100-04). Mini-prep (alkaline lysis) DNA preparations are not recommended for MultiSite Gateway® cloning reactions.
- DNA cannot be quantitated by UV absorbance due to contaminating RNA and nucleotides, estimate concentration by gel electrophoresis (e.g., DNA Mass Ladder, Cat. no. 10068-013 or 10496-016).
- For LR reactions, supercoiled entry vectors and destination vectors provide efficient substrates.
- For large (>10 kb) entry clones or destination vectors, linearizing the entry clone or destination vector may increase the efficiency by up to two fold.
Materials
Components:
Gateway® LR Clonase™ II Plus Enzyme Mix (40 μl)
2 μg/μl Proteinase K Solution (40 μl)
Storage:
Store Gateway® LR Clonase™ II Plus at -20°C (in a non-frost-free freezer) for up to 6 months. For long term storage, store at -80°C.
Reaction Setup
Reaction Setup
For multi-fragment (i.e. 2-, 3-, or 4-fragment recombination) reactions, use an equimolar amount of each entry clone. We recommend 10 fmol of each entry clone and 10 fmol of DEST vector per 5 μl reaction.
Plasmid mixture:
Add the following components to a 1.5-ml microcentrifuge tube at room temperature and mix. Use this reaction mixture in the following procedure.
- Entry clones (10 fmoles)*
- Destination vector (10 fmoles)
- at least 1 μl 1X TE buffer, pH 8.0 for a total volume of 4µl
*All entry clones (two, three or four, depending on the type of reaction) must be included. The total of all plasmids and buffer combined should not exceed 4 μl.
Use a calculator to determine the amount of each plasmid to add to the reaction. For example, you would need to add 29 ng of a 4.25 kb plasmid to get 10 fmol. https://www.promega.com/resources/tools/biomath/
Procedure
Procedure
16h 12m
16h 12m
Remove LR Clonase™ II Plus enzyme mix from freezer and thaw on ice for about 00:02:00 . Vortex the enzyme mix briefly twice (2 seconds each).
2m
To each MultiSite or MultiSite Pro LR reaction mixture, add 1 μl of LR Clonase™ II Plus and mix well by vortexing briefly twice. Microcentrifuge briefly.
Return enzyme mix to freezer immediately after use. The enzyme mix can be stored at -20°C for up to 6 months or at -80°C for long-term storage.
Incubate recombination reaction at 25°C for 16:00:00 .
16h
Add 0.5 μl of the Proteinase K solution to each sample to terminate the
reaction. Vortex briefly. Incubate samples at 37°C for 00:10:00 .
10m
Transformation
Transformation
16h 12m
16h 12m
For 2- or 3-fragment recombination reactions, add 2-3 μl to 50 μl of Mix & Go Chemically Competent E. coli and incubate on ice for 5-30 minutes.
For 4-fragment recombination reactions, add 4-5 μl to 50 μl of Mix & Go Chemically Competent E. coli and incubate on ice for 5-30 minutes.
Add 200 μl of SOC medium and incubate at 37°C for 1-1.5 hour with shaking at 225-250 RPM. Can be omitted with Ampicillin resistance.
Plate entire reaction on selective media.
Note
Typical Numbers of Colonies (per 10 μl reaction):
2-fragment recombination reaction: 2,000-15,000
3-fragment recombination reaction: 1,000-5,000
4-fragment recombination reaction: 50-500
Note
Some labs have found that clear colonies contain the correct clone >99% of the time, while opaque colonies never contain the correct clone. A reaction that has worked well will have a clear to opaque colony ratio of at least 3:1. However, as long as clear colonies can be identified, the correct clone will be isolated. Clones can be tested via restriction digest or colony PCR.