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Team:Paris Bettencourt/Notebook/Trojan Horse/Saturday 14th September.html
From 2013.igem.org
Trojan Horse
ASDF14th September
Place your twit here
Cloning two sRNA on the same phagemid
P009 (litmus Kan) + sRNA Cm :
P027 (litmus Cm) + sRNA Kan :
Inserts
sRNA Cm : 35 ng/uL Final (16*35/20) = 28 ng/uL
sRNA Kan : 16 ng/uL Final (16*16/20) = 12,8 ng/uL
|
Reagent |
Volume |
|
Purified PCR Product |
16 ul |
|
H2O |
0 ul |
|
10x FastDigest Buffer |
2 ul |
|
XbaI |
1 ul |
|
Pst1 |
1 ul |
|
Total Volume |
20.0 ul |
Vectors
Litmus Kan : 350 ng /uL Final (3*350/20) = 52,5 75 ng/ul
Litmus Cm : 750 ng /ul Final (2*750/20) = 75 ng/uL
|
Reagent |
Volume |
|
Purified PCR Product |
3 |
|
H2O |
13 ul |
|
10x FastDigest Buffer |
2 ul |
|
Pst1 |
1 ul |
|
SpeI |
1 ul |
|
Total Volume |
20.0 ul |
Ligation sRNA into biobrick vector
Litmus Kan + sRNA anti Cm
masse insert = 10*419/4000*100 = 110 ng
Vector : 3 uL
Insert : 4 uL
Buffer : 2 uL
T4 DNA ligase : 1 uL
H2O : 10uL
Litmus Cm + sRNA anti Kan
masse insert = 10*419/4000*100 = 110 ng
Vector : 1,5 uL
Insert : 10 uL
Buffer : 2 uL
T4 DNA ligase : 1 uL
H2O : 5,5uL
Let incubate at 22°C for 30 min
Launch cultures :
Glycerol of the different biobricks : sT040 ; sT041 ; sT042 sT043 ; sT044 ; sT045 ; sT046 ; sT047 on Cm
MG F+ + pCola sT027 on Kan :
MGZ1, F+ Litmus-GFP-sRNA antiKan + M13KO7 Helper col : sT022 sT023 sT024 on Kan Amp
