Indiana/1 July 2009

From 2009.igem.org

(Difference between revisions)
(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...)
 
(4 intermediate revisions not shown)
Line 1: Line 1:
Construction of plant plasmid pCB302 to fit iGEM standards.
Construction of plant plasmid pCB302 to fit iGEM standards.
-
Step 1
 
-
Remove Xba1 and Spe1 sites from pCD302
+
'''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 22: 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
 +
 +
 +
 +
 +
 +
 +
 +
'''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

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

Retrieved from "http://2009.igem.org/Indiana/1_July_2009"