Thermo Fisher Scientific Gateway LR Clonase II Plus User guide

Brand
Thermo Fisher Scientific
Model
Gateway LR Clonase II Plus
Type
User guide
Gateway™ LR Clonase™ II Plus Enzyme Mix
Catalog Numbers 12538120, 12538200
Pub. No. MAN0001087 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The Invitrogen™ 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.
The Gateway™ LR Clonase™ II Plus Enzyme Mix is a proprietary enzyme formulation specifically optimized for both single- and multiple-
fragment cloning. The Gateway™ LR Clonase™ II Plus Enzyme Mix contains the following in a single mix of enzyme and reaction buer,
ensuring enzyme stability and ease-of-use with few pipetting steps:
• Bacteriophage lambda recombination proteins Integrase (Int) and Excisionase (Xis)
•E. coli-encoded protein Integration Host Factor (IHF)
• Reaction buer
Contents and storage
Contents Cat. No. 12538120
(20 rxns)
Cat. No. 12538200
(100 rxns) Storage
Gateway™ LR Clonase™ II Plus Enzyme Mix 40 μL 200 μL
−80°C.
Proteinase K Solution (2 μg/μL) 40 μL 200 μL
Procedural guidelines
• We recommend using plasmid DNA purified with the
PureLink™ HiPure Plasmid Midiprep Kit (Cat. No. K210004).
Mini-prep (alkaline lysis) DNA preparations are not
recommended for MultiSite Gateway™ Pro cloning reactions.
DNA cannot be quantitated by UV absorbance because of
contaminating RNA and nucleotides. Estimate concentration
by gel electrophoresis, e.g., Low DNA Mass Ladder
(Cat. No. 10068013) or High DNA Mass Ladder
(Cat. No. 10496016).
• For LR reactions, supercoiled entry vectors and destination
vectors provide ecient substrates.
• For large (>10 kb) entry clones or destination vectors,
linearizing the entry clone or destination vector may increase
the eciency by up to 2‑fold.
• To convert fmoles to nanograms (ng):
ng = (x fmol)(N)(660 fg/fmol)(1 ng/106 fg)
where:
– N is the size of the DNA in base pairs
–x is the number of fmoles
Perform the LR reaction
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 20 fmol of DEST
vector per 10‑μL reaction.
Note: If you are using the MultiSite Gateway™ Pro Technology, you
can recombine up to four entry clones into any pDEST vector that
contains att R1 and att R2 sites.
1. Add the following components to a 1.5‑mL microcentrifuge
tube at room temperature, then mix:
Component Volume
Entry clones (10 fmoles) 1 µL−7 μL total[1]
Destination vector (20 fmoles) 1 μL
1X TE Buer, pH 8.0 to 8 μL
[1] All entry clones (two, three, or four, depending on the type of reaction) must
be included. The combined total of all entry clones should not exceed 7 μL.
2. Thaw on ice the LR Clonase™ II Plus Enzyme Mix for
~2 minutes. Briefly vortex the enzyme mix twice (2 seconds
each time).
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.
3. Add 2 μL of LR Clonase™ II Plus Enzyme Mix to each
MultiSite Gateway™ Pro LR reaction, then briefly vortex twice
to mix well. Briefly microcentrifuge.
4. Return the LR Clonase™ II Plus Enzyme Mix to −80°C
storage immediately after use.
5. Incubate the reactions at 25°C for 16 hours.
6. Add 1 μL of the Proteinase K Solution to each sample to
terminate the reaction. Briefly vortex. Incubate the samples
at 37°C for 10 minutes.
Transform competent cells
1. Add One Shot™ Mach1™ T1 Phage-Resistant Chemically
Competent E. coli (Cat. No. C862003) to each reaction as
follows:
Recombination reaction Chemically Competent E. coli
2-fragment 2 μL−50 μL
3-fragment 2 μL−50 μL
4-fragment 4 μL−50 μL
2. Incubate on ice for 30 minutes.
3. Heat-shock the cells by incubating at 42°C for 30 seconds.
4. Immediately transfer the tubes to ice for 2 minutes.
5. Add 250 μL of S.O.C. Medium, then incubate at 37°C for
1 hour with shaking at 225 rpm.
6. For 2-fragment recombination reactions:
a. Dilute 1:10 in S.O.C. Medium.
b. Plate 50 μL and 100 μL of each
transformation on prewarmed LB plates that contain
50 μg/mL−100 μg/mL of antibiotic of choice, invert,
then incubate overnight at 37°C.
7. For 3‑fragment recombination reactions: Plate 50 μL and
100 μL of each transformation as in substep 6b.
8. For 4-fragment recombination reactions:
a. Spin at 6,000 rpm, remove 180 μL of supernatant, then
gently resuspend the pellet in the residual media.
b. Plate the entire reaction as in substep 6b.
Expected results
Recombination reaction No. of colonies
(per 10‑μL reaction)
2-fragment 2,000−15,000
3-fragment 1,000−5,000
4-fragment 50−500
Next steps
For instructions on expressing the recombinant protein in the
appropriate system, see the guide for the destination vector that
you are using.
For more information, see the Gateway™ and MultiSite Gateway™
Pro Technology documentation, available at thermofisher.com.
Quality control
Gateway™ LR Clonase™ II Plus Enzyme Mix is functionally tested
in a 16-hour reaction to combine 4 fragments into one DEST
vector, followed by a transformation assay.
MultiSite Gateway™ and MultiSite Gateway™ Pro
MultiSite Gateway™ and MultiSite Gateway™ Pro are extensions
of the Gateway™ site-specific recombinational cloning technology,
which is based on the recombination properties of bacteriophage
lambda. MultiSite Gateway™ allows cloning of exactly three DNA
fragments into pDEST R4R3, while MultiSite Gateway™ Pro allows
cloning of two, three, or four DNA fragments in a defined order
and orientation into any pDEST vector containing attR1 and
attR2 sites. Both systems provide a rapid and ecient way
to recombine DNA elements into vector systems for functional
analysis and protein expression. The LR recombination reaction
occurs between two specific attachment sites (attL and attR)
on the entry clones and the destination vector, allowing the
recombination of fragments into the destination vector. The
reaction is mediated by Gateway™ LR Clonase™ II Plus Enzyme
Mix.
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their
products as set forth in the Life Technologies' General Terms
and Conditions of Sale at www.thermofisher.com/us/en/home/
global/terms-and-conditions.html. If you have any questions,
please contact Life Technologies at www.thermofisher.com/
support.
2 Gateway™ LR Clonase™ II Plus Enzyme Mix User Guide
Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0001087
Revision Date Description
A.0 26 Jan 2022 Changed storage temperature to -80C. Updated format, style, and legal text.
— 11 July 2006 Baseline for this revision history
Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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26 January 2022
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Thermo Fisher Scientific Gateway LR Clonase II Plus User guide

Brand
Thermo Fisher Scientific
Model
Gateway LR Clonase II Plus
Type
User guide
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