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.

thermoﬁsher.com/support | thermoﬁsher.com/askaquestion

thermoﬁsher.com

26 January 2022

Thermo Fisher Scientific Gateway LR Clonase II Plus User guide

Brand: Thermo Fisher Scientific

Model: Gateway LR Clonase II Plus

Type: User guide

Download PDF

report

Thermo Fisher Scientific Gateway LR Clonase II Plus User guide

Thermo Fisher Scientific Gateway LR Clonase II Plus User guide

Related papers

Other documents