Team:Paris Bettencourt/Notebook/Trojan Horse/Saturday 14th September.html

From 2013.igem.org

Trojan Horse

14th 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