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Indiana/1 July 2009
From 2009.igem.org
Construction of plant plasmid pCB302 to fit iGEM standards.
Step 1
Remove Xba1 and Spe1 sites from pCD302
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
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
