**8.4. MultiSite Gateway LR reaction**

Destination vector (20 fmol/μl; Invitrogen): 0.5μl

attB1/attB5r entry clone (10 fmol/μl): 0.5μl

attB5/attB4 entry clone (10 fmol/μl): 0.5μl

attB4r/attB3r entry clone (10 fmol/μl): 0.5μl

attB3/attB2 entry clone (10 fmol/μl): 0.5μl

TE buffer: 1.5μl

LR Clonase II Plus enzyme mix (Invitrogen): 1μl

(Note: LR Clonase enzyme and LR Clonase II enzyme mix, which are used for standard single fragment cloning, cannot be used for cloning multiple fragments.)-

Incubate at 25°C for 1–2days.-

Add 0.5μl Proteinase K solution (20mg/ml; Invitrogen) to the reaction mixture. Incubate at 37°C for 10min.-

Transform the reaction mixture into Mach T1 competent cells (Invitrogen) by the heat-shock method. Spread the transformed cells on LB agar plates containing appropriate antibiotics and incubate them at 37°C overnight. The plasmid DNA is purified from the colonies on the LB agar plates and used as the donor DNA after verification.-

(Notes: Mach T1 competent cells were more suitable for cloning multiple fragments than DH5α competent cells.)-
