TITLE: LR Clonase Reaction for Multisite Gateway Cloning
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Gateway® LR Clonase™ II Plus</span><span></span></div><div class = "text-block"><div class = "justify" style = "text-align:justify">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.</div><style>
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[STEPS]
?. [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]
Remove LR Clonase™ II Plus enzyme mix from freezer and thaw on ice for about . Vortex the enzyme mix briefly twice (2 seconds each).
?. [Procedure]
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.
?. [Procedure]
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.
?. [Procedure]
Incubate recombination reaction at 25°C for .
?. [Transformation]
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.
?. [Procedure]
Add 0.5 μl of the Proteinase K solution to each sample to terminate thereaction. Vortex briefly. Incubate samples at 37°C for .
?. [Transformation]
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.
?. [Transformation]
Plate entire reaction on selective media.
Typical Numbers of Colonies (per 10 μl reaction):2-fragment recombination reaction: 2,000-15,0003-fragment recombination reaction: 1,000-5,0004-fragment recombination reaction: 50-500
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.
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