MultiSite Gateway Cloning

MultiSite Gateway Cloning Protocol

2-fragment recombination

Element 1 attB1, attB5r
Element 2 attB5, attB2

forward PCR primers:

attB1 5'-GGGG-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-TNN--(template-specific sequence)-3'
attB5 5'-GGGG-ACA-ACT-TTG-TAT-ACA-AAA-GTT-GNN--(template-specific sequence)-3'

reverse PCR primers
attB2 5'-GGGG-AC-CAC-TTT-GTA-CAA-GAA-AGC-TGG-GTA--(template-specific sequence)-3'
attB5r 5'-GGGG AC AAC TTT TGT ATA CAA AGT TGT--(template-specific sequence)-3'

3- fragment recombination

Element 1 attB1, attB4
Element 2 attB4r, attB3r
Element 3 attB3, attB2

forward PCR primers

attB1 5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGC TNN--(template-specific sequence)-3'
attB4r 5'-GGGG ACA ACT TTT CTA TAC AAA GTT GNN--(template-specific sequence)-3'
attB3 5'-GGGG ACA ACT TTG TAT AAT AAA GTT GNN--(template-specific sequence)-3'
reverse PCR primers
attB4 5'-GGGG AC AAC TTT GTA TAG AAA AGT TGG GTG--(template-specific sequence)-3'
attB3r 5'-GGGG AC AAC TTT ATT ATA CAA AGT TGT--(template-specific sequence)-3'
attB2 5'-GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA--(template-specific sequence)-3'

4- fragment recombination

Element 1 attB1, attB5r
Element 2 attB5, attB4
Element 3 attB4r, attB3r
Element 4 attB3, attB2
forward PCR primers
attB1 5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGC TNN--(template-specific sequence)-3'
attB5 5'-GGGG ACA ACT TTG TAT ACA AAA GTT GNN--(template-specific sequence)-3'
attB4r 5'-GGGG ACA ACT TTT CTA TAC AAA GTT GNN--(template-specific sequence)-3'
attB3 5'-GGGG ACA ACT TTG TAT AAT AAA GTT GNN--(template-specific sequence)-3'

reverse PCR primers
attB5r 5'-GGGG AC AAC TTT TGT ATA CAA AGT TGT--(template-specific sequence)-3'
attB4 5'-GGGG AC AAC TTT GTA TAG AAA AGT TGG GTG--(template-specific sequence)-3'
attB3r 5'-GGGG AC AAC TTT ATT ATA CAA AGT TGT--(template-specific sequence)-3'
attB2 5'-GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA--(template-specific sequence)-3'

Note:
Template- or gene-specific sequences: 18-25 bp
NN cannot be AA, AG, or GA
The primer must include two additional nucleotides to maintain the proper reading frame for N- or C- fusion tags, and remove stop codon.

BP Reaction

1. Add:
attB PCR product (15-150 ng) 1-7 ÎŒl
pDONR vector (150 ng/ÎŒl) 1 ÎŒl
1X TE Buffer, pH 8.0, to 8 ÎŒl

2. Remove the BP ClonaseTM II enzyme mix from -20°C or -80°C and thaw on ice (~ 2 minutes).

3. Vortex the BP ClonaseTM II enzyme mix briefly twice (2 seconds each time).

4. Add 2 ÎŒl of BP ClonaseTM II enzyme mix. Mix well by vortexing briefly twice (2 seconds each time).
Reminder: Return BP ClonaseTM II enzyme mix to -20°C or -80°C immediately after use.

5. Incubate reactions at 25°C for 1 hour.
The length of the recombination reaction can be extended up to 18 hours.
An overnight incubation typically yields 5-10 times more colonies than 1 hour incubation.
For large PCR products (≥5 kb), longer incubations (i.e. overnight incubation) will increase the yield of colonies and are recommended.

6. Add 1 ÎŒl of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37°C.

7. Proceed to Transforming One Shot® Mach1TM T1R Competent Cells, next page.

8. Transform One Shot® Mach1TM T1R competent cells

LR Reaction

1. Add:
Entry clones (10 fmoles each) 1-7 ÎŒl
Destination vector (20 fmoles) 1 ÎŒl
1X TE Buffer, pH 8.0, to 8 ÎŒl

2. Remove the LR ClonaseTM II Plus enzyme mix from- 20°C or -80°C and thaw on ice (~ 2 minutes).

3. Vortex the LR ClonaseTM II Plus enzyme mix briefly twice (2 seconds each time).

4. To each sample above, add 2 ÎŒl of LR ClonaseTM Plus enzyme mix. Mix well by vortexing briefly twice (2 seconds each time).

5. Return LR ClonaseTM II Plus enzyme mix to –20 °C or -80°C 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.

6. Incubate reactions at 25°C for 16 hours.

7. Add 1 ÎŒl of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37°C.

8. Transforming One Shot® Mach1TM T1R competent cells