Team:KIT-Kyoto/kazukokekokko

BP reaction by Invitrogen gateway system

1. PCR using primers containing the attB sequence.
2. Purify PCR product.
3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

attB PCR product	75 ng/reaction (1-7 μL)
pDONR vector	150ng/reaction (1 μL)
TE Buffer	to 8 μL

4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing and spin them down.
5. Incubate reaction at 25˚C for more than 1 hour.
6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
7. Incubate at 37˚C for 10 minutes.

LR reaction by Invitrogen gateway system

1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

Entry clones	50-150 ng/reaction (1-7 μL)
Destination vector	150ng/reaction (1 μL)
TE Buffer	to 8 − 9 μL

2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above and mix well by vortexing and spin down.
3. Incubate reaction at 25˚C for 16 hours.
4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.

5. Incubate at 37˚C for 10 minutes.