Indiana/1 July 2009

Construction of plant plasmid pCB302 to fit iGEM standards.

Remove Xba1 and Spe1 sites from pCB302

1 uL pCB302 @ ~10ng/ul

2 uL NEB buffer 4

.3 uL Xba1

.3 uL Spe1

16.4 uL ddH2O

incubate 1 hour @ 37 C

Ligate plasmid back together

following incubation add the following the reaction:

4 uL ligase buffer

1 uL ligage

15 uL ddH2O

incubate at 16 C for ~3 hours

Transform newly modified plasmid into E. coli

5 uL of reaction mixture into chemically competent E. coli

keep on ice for 15 min

heat shock @ 37 C for 2 minutes

add to 500 uL LB, shake at 37 C for 30-60 minutes

spin down and resuspend in a smaller volume (50-100uL)

plated on kanmyacin plates

Pull Parts from Kit

J45119 - wintergreen

J45199 - banana

psB1AT3 - plasmid backbone

1) add 15 uL ddH2O to proper well on plate

2) let sit for ~5 minutes

3) transfer to tube for storage

4) transform using 1uL of part