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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 | ||
| + | |||
2 uL NEB buffer 4 | 2 uL NEB buffer 4 | ||
| + | |||
.3 uL Xba1 | .3 uL Xba1 | ||
| + | |||
.3 uL Spe1 | .3 uL Spe1 | ||
| + | |||
16.4 uL ddH2O | 16.4 uL ddH2O | ||
| + | |||
incubate 1 hour @ 37 C | incubate 1 hour @ 37 C | ||
| + | |||
| + | |||
| + | '''Ligate plasmid back together''' | ||
following incubation add the following the reaction: | following incubation add the following the reaction: | ||
4 uL ligase buffer | 4 uL ligase buffer | ||
| + | |||
1 uL ligage | 1 uL ligage | ||
| + | |||
15 uL ddH2O | 15 uL ddH2O | ||
| Line 21: | Line 31: | ||
| - | Transform newly modified plasmid into E. coli | + | |
| + | |||
| + | |||
| + | '''Transform newly modified plasmid into E. coli''' | ||
| + | |||
5 uL of reaction mixture into chemically competent E. coli | 5 uL of reaction mixture into chemically competent E. coli | ||
keep on ice for 15 min | keep on ice for 15 min | ||
| + | |||
heat shock @ 37 C for 2 minutes | heat shock @ 37 C for 2 minutes | ||
| + | |||
add to 500 uL LB, shake at 37 C for 30-60 minutes | add to 500 uL LB, shake at 37 C for 30-60 minutes | ||
| + | |||
spin down and resuspend in a smaller volume (50-100uL) | spin down and resuspend in a smaller volume (50-100uL) | ||
| + | |||
plated on kanmyacin plates | plated on kanmyacin plates | ||
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| - | Pull Parts from Kit | + | '''Pull Parts from Kit''' |
| + | |||
J45119 - wintergreen | J45119 - wintergreen | ||
| + | |||
J45199 - banana | J45199 - banana | ||
| + | |||
psB1AT3 - plasmid backbone | psB1AT3 - plasmid backbone | ||
1) add 15 uL ddH2O to proper well on plate | 1) add 15 uL ddH2O to proper well on plate | ||
| + | |||
2) let sit for ~5 minutes | 2) let sit for ~5 minutes | ||
| + | |||
3) transfer to tube for storage | 3) transfer to tube for storage | ||
| + | |||
4) transform using 1uL of part | 4) transform using 1uL of part | ||
Latest revision as of 19:05, 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
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
