**8.3. BP reaction**

pDONR entry vector (150ng/μl; Invitrogen): 0.25μl

BP Clonase II enzyme mix (Invitrogen): 0.5μl

PCR product: 1–1.75μl (10–100 ng)

TE buffer: to final reaction volume of 2.5μl

Incubate at 25°C for 60min (for attB4r/attB3r and attB3/attB2 fragments) or overnight (for attB1/attB5r and attB5/attB4 fragments).-

(Note: the cloning efficiencies of the attB1/attB5r and attB5/attB4 fragments were low. A longer incubation time in the BP reaction improved the cloning efficiency.)-

Add 0.25μl Proteinase K solution (20mg/ml; Invitrogen) to the reaction mixture. Incubate at 37°C for 10min.-

Transform the reaction mixture into DH5α competent cells by the heat-shock method. Spread the transformed cells on an LB agar plate containing 20μg/ml of kanamycin and incubate it at 37°C overnight. The plasmidsare purified from each colony on the LB agar plate and are verified by DNA sequencing.-
