Gateway Cloning Protocols

Step 1—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 2—Perform the TOPO Cloning Reaction    

1. Set up one of the following Invitrogen TOPO Cloning reactions using the reagents in the order shown. For electroporation, dilute Salt Solution 4-fold to prepare Dilute Salt Solution.
   

   Reagent	Chemical reaction	Electroporation
   Fresh PCR product	0.5 to 4 µl	0.5 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 volume	6 µl	6 µl

2. Mix gently and incubate for 5 minutes at room temperature.
3. Place on ice and proceed to transform Invitrogen One Shot chemically competent E. coli, below

Step 3—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 5–30 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.

BP reaction

Creating a Gateway entry clone from an attB-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 Invitrogen 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 Invitrogen One Shot OmniMAX 2 T1 Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes. Heat-shock cells by incubating at 42°C for 30 seconds. Add 250 µl of S.O.C. Medium and incubate at 37°C for 1 hour with shaking. Plate 20 µl and 100 µl of each transformation onto selective plates. Note: Any competent cells with a transformation efficiency of >1.0 × 10 8 transformants/µg may be used.
2. Transform 1 µl of pUC19 DNA (10 ng/ml) into 50 µl of One Shot OmniMAX 2 T1 Phage-Resistant Cells as described above. Plate 20 µl and 100 µl on LB plates containing 100 µg/ml kanamycin, or the appropriate selection marker for your donor vector.

 
Expected results
An efficient BP recombination reaction will produce >1500 colonies if the entire BP reaction is transformed and plated. 

LR reaction

Transferring your gene from a Gateway entry clone to destination vector 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:
    Entry clone (50-150 ng) 1–7 µl
    Destination vector (150 ng/µl) 1 µl
    TE buffer, pH 8.0 to 8 µl
2. Thaw on ice the Invitrogen LR Clonase  II enzyme mix for about 2 minutes. Vortex the LR Clonase II enzyme mix briefly twice (2 seconds each time).
3. To each sample (Step 1, above), add 2 µl of LR Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.
4. Return LR 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
Follow the protocol as indicated for the BP reaction, except use the appropriate selection marker for the LB plates suited to your destination vector (typically 100 µg/ml ampicillin).

Expected results
An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated. 

One tube format

If you want to transfer your attB-flanked PCR product directly into an expression clone, you can easily combine the BP and LR reactions using the following protocol. This will potentially eliminate the transformation and DNA isolation of the Gateway entry clone.

1. In a 1.5 ml microcentrifuge tube, prepare the following 15 µl BP reaction:
    attB DNA (50-100 ng) 1.0–5.0 µl
    attP DNA (Invitrogen pDONR vector, 150 ng/µl) 1.3 µl
    BP Clonase II enzyme mix 3.0 µl
    TE Buffer, pH 8.0 add to a final volume of 15 µl
2. Mix well by vortexing briefly and incubate at 25°C for 4 hours.
   Note: Depending on your needs, the length of the recombination reaction can be extended up to 20 hours. An overnight incubation typically yields 5 times more colonies than a 1 hour incubation. Longer incubation times are recommended for large plasmids (=10 kb) and PCR products (=5 kb).
3. Remove 5 µl of the reaction to a separate tube and use this aliquot to assess the efficiency of the BP reaction (see below).
4. To the remaining 10 µl reaction, add:
    Destination vector (150 ng/µl) 2.0 µl
    LR Clonase II enzyme mix 3.0 µl
    Final volume 15 µl
5. Mix well by vortexing briefly and incubate at 25°C for 2 hours.
   Note: Depending on your needs, the length of the recombination reaction can be extended up to 18 hours.
6. Add 2 µl of proteinase K solution. Incubate at 37°C for 10 minutes.
7. Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction.
8. Plate on LB plates containing the appropriate antibiotic to select for expression clones.
   

Assessing the efficiency of the BP reaction

1. To the 5µl aliquot obtained from “One-Tube” Protocol, Step 3, above, add 0.5 µl of proteinase K solution. Incubate at 37°C for 10 minutes.
2. Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction. Plate on LB plates containing the appropriate antibiotic to select for entry clones. 

Gateway vector conversion

Converting your favorite set of cloning vectors to Gateway Technology is a fairly straightforward protocol, and will ultimately allow you to streamline your cloning and expression process.

To convert your cloning vector to a Gateway destination vector, you will:

1. Choose the appropriate reading frame cassette to use depending on your needs.
2. Linearize the vector you wish to convert with a restriction enzyme of choice. If you use a restriction enzyme that generates an overhang, you will need to blunt the ends.
3. Remove the 5' phosphates from the vector using calf intestinal alkaline phosphatase.
4. Ligate the reading frame cassette into your vector using T4 DNA ligase.
5. Transform the ligation reaction into One Shot ccdB Survival Competent E. coli and select for transformants.
6. Analyze transformants.