Gateway技术操作指南

Gateway® Cloning Protocols

The Basics of Gateway® Reaction

∙BP reaction—to create a Gateway® entry clone

∙LR reaction—to create a Gateway® expression clone

∙One tube format—to create a Gateway® expression clone from a PCR product

∙Gateway® Vector Conversion—converting your favorite cloning vectors to Gateway® Technology

TOPO® TA Cloning - To Create a Gateway® Entry Clone

Step One - Produce PCR product

Produce PCR products using Taq polymerase and your own protocol. End

the PCR reaction with a final 7 to 30 minute extension step.

Step Two - Perform the TOPO® Cloning Reaction

1. Set up one of the following TOPO® Cloning reactions using the

reagents in the order shown. For electroporation, dilute Salt

Solution 4-fold to prepare Dilute Salt Solution.

Reagent Chemical Txn Electroporation

Fresh PCR product0.5 to 4 µl0.5 to 4 µl

Salt Solution 1 µl--

Dilute Salt

Solution

-- 1 µl

Sterile Water to a final volume of 5

µl

to a final volume of 5

µl

TOPO® Vector 1 µl 1 µl

Total volume6µl6µl

2. Mix gently and incubate for 5 minutes at room temperature.

3. Place on ice and proceed to transform One Shot® chemically competent E. coli, below

Step Three - Transform One Shot® Chemically Competent E. coli

1. For each transformation, thaw one vial of One Shot® E. coli cells on ice.

2. Add 2 µl of the TOPO® Cloning reaction into a vial of One Shot® chemically competent E. coli and mix gently.

3. Incubate on ice for 5 to 30 minutes.

4. Heat-shock the cells for 30 seconds at 42°C without shaking. Immediately

transfer the tube to ice.

5. Add 250 µl of room temperature S.O.C. Medium.

6. Incubate at 37°C for 1 hour with shaking.

7. Spread 10-50 µl of bacterial culture on a prewarmed LB agar plate containing

100 µg/ml spectinomycin, and incubate overnight at 37°C.

The Basics of Gateway® Reactions

BP Reaction

Creating a Gateway® entry clone from an att B-flanked PCR product is an easy 1 hour reaction. See below for an overview of the set-up. For more detailed information, refer to the manual.

1.Add the following components to a 1.5 ml tube at room

temperature and mix:

attB-PCR product (=10 ng/µl; final amount ~15-150 ng) 1-7 µl

Donor vector (150 ng/µl) 1 µl

TE buffer, pH 8.0 to 8 µl

2.Thaw on ice the BP Clonase™ II enzyme mix for about 2 minutes.

Vortex the BP Clonase™ II enzyme mix briefly twice (2 seconds

each time).

3.To each sample (Step 1, above), add 2 µl of BP Clonase™ II

enzyme mix to the reaction and mix well by vortexing briefly

twice. Microcentrifuge briefly.

4.Return BP Clonase™ II enzyme mix to -20°C or -80°C storage.

5.Incubate reactions at 25°C for 1 hour.

6.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.

Transformation

1.Transform 1 µl of each BP reaction into 50 µl of One Shot ®

OmniMAX ™ 2 T1 Phage-Resistant Cells (Catalog no. C8540-03).