Team:Paris Bettencourt/Notebook/Trojan Horse/Wednesday 18th September.html

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Trojan Horse

ASDF

18^th 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;

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      <em>Miniprep biobricks</em>