Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 5th September.html

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Trojan Horse

5th September


Confirmation of silencing (CmR)

Cloning biobricks : ie clone sRNAs (Cm, Kan, lacZ) in pB1C3

Reagent

Volume

Purified PCR Product

16 ul

H2O

0 ul

10x FastDigest Buffer

2 ul

FastDigest Enzyme 1

1 ul

FastDigest Enzyme 2

1 ul

Total Volume

20.0 ul

Digest for 0.5 - 1 hours at 37 °C with shaking

Ligation sRNA into biobrick vector

masse insert = 10*419/2000*100 = 210 ng

Vector : 5 uL

Insert : 10 uL

Buffer : 2 uL

T4 DNA ligase : 1 uL

H2O : 2uL

Let incubate at 22°C for 30 min

Transformation in NEB turbo chemical competent . Plate on Cm

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