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Indiana/1 July 2009
From 2009.igem.org
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| - | Remove Xba1 and Spe1 sites from | + | Remove Xba1 and Spe1 sites from pCB302 |
1 uL pCB302 @ ~10ng/ul | 1 uL pCB302 @ ~10ng/ul | ||
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Pull Parts from Kit: | Pull Parts from Kit: | ||
| + | |||
J45119 - wintergreen | J45119 - wintergreen | ||
Revision as of 19:03, 19 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
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
