Indiana/1 July 2009

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(New page: 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 i...)
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Revision as of 18:55, 19 July 2009

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