Team:KIT-Kyoto/kazukokekokko

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Revision as of 14:49, 14 September 2012 by Kazuko (Talk | contribs)







BP reaction by invitrogen gateway system


1. PCR using promers containing the attB sequence.
*attB sequence:
2. Purification PCR products.
3. Prepare vials in 1.5mL tube according to the following components.

attB DNA sample75ng/reaction
pDONR vector150ng/reaction
TE Bufferup
Total8uL or 9uL

4. Dissolve BP Clonase Ⅱ enzyme mix on ice.
5. Add BP Clonase Ⅱ enzyme mix (2uL or 1uL) to vials, vortex and spin them down.
6. Incubate vials at 25℃ for more than an hour.
7. Add Proteinase K solutionto vials (1uL) and vortex shortly.
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 sample50-150ng/reaction
Destination vector150ng/reaction
TE Bufferup
Total8uL or 9uL

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


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