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Team:Paris Bettencourt/Notebook/Trojan Horse/Sunday 15th September.html
From 2013.igem.org
Trojan Horse
ASDF15th September
Exp Silencing Kan
- Launch O/N culture of producing phages cells ; centrifuge ; take supernatant and filter it.
-diluting 100x O/N of MGZ1, F+ -pCola in LB-Kan (25 ug/ul)
- At OD 0,7 :. Put 900 uL of cells in each tube. Add in tubes respectively : nothing, 100uL phages of litmusGFP-LacZ1, litmusGFP-Kan1 & litmusGFP-Kan2
-2 replicates for each condition. Plate each Dilution 2 times to limit the variability
Non infected cells (1,2)
Cells infected with anti lacZ (1,2)
Cells infected with anti KanA (1,2)
Cells infected with anti KanB (1,2,)
- Incubate 70 minutes at 37°C
- Serial Dilute every tube until 10-5. Plate 100ul for each tube according to this dilution table
|
LB |
Kan 25 ug/ul |
Kan 250ug/ul |
Kan 500 ug/ul |
|
|
non infected 1 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-4/3 |
|
non infected 2 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-4/3 |
|
sRNA lacZ 1 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-3/2 |
|
sRNA lacZ 2 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-3/2 |
|
sRNA Kan A 1 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-3/2 |
|
sRNA Kan A 2 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-3/2 |
|
sRNA Kan B 1 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-3/2 |
|
sRNA Kan B 2 |
10^-5/4 |
10^-4/3 |
10^-4/3 |
10^-3/2 |
Miniprep biobricks
Cm1 / Cm2 / Kan 1 / Kan 2 / Lac1 / Lac2
Cloning 2 sRNA on the same phage : 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
Transformation in chemical competent 10G cells : plate on Amp;
