Team:KIT-Kyoto/kazukokekokko

BP reaction by invitrogen gateway system

1. PCR using primers containing the attB sequence.
2.Purify PCR product.
3. Prepare vials in 1.5mL tube according to the following components.

attB DNA sample	75ng/reaction
pDONR vector	150ng/reaction
TE Buffer	up
Total	8uL or 9uL

4. Melt BP Clonase Ⅱ enzyme mix on ice.
5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to tube, vortex and spin them down.
6. Incubate vials at 25℃ for more than an hour.
7. Add Proteinase K solutionton (1uL) vials and vortex briefly.
8. Incubate at 37℃ for 10 minutes.

LR reaction by invitrogen gateway system

1. Control vials in 1.5mL tube according to the following components.

BP DNA sample	50-150ng/reaction
Destination vector	150ng/reaction
TE Buffer	up
Total	8uL or 9uL

2.Melt LR Clonase Ⅱ enzyme mix on ice
3. Add LR Clonase Ⅱ enzyme mix to tube (2uL or 1uL), votex and spin down.
4. Incubate vials at 25℃ for more than an hour.
5. Add Proteinase K solution (1μl) to vials and vortex it briefly.
6. Incubate at 37℃ for 30 minutes.