Team:Paris Bettencourt/Notebook/Trojan Horse/Tuesday 27th August.html

Trojan Horse

ASDF

27^th August

Place your twit here

Building anti LacZ weapons:

Gibson assembly of litmus (sT005) and sRNA_LacZ

Digestion vector : 56 ng/ul

Digestion insert : 9,6

masse insert = 5*376/3626*50 = 26 ng

Vector : 1 uL

Insert : 3 uL

Master Mix : 5 uL

H2O : 1

Heat shock following Gibson assembly kit protocole.

Biobrick assembly of sRNA_LacZ in litmus-GFP and pUC18

Using digested vectors (litmus-GFP and pUC18) from 26/08

Digestion of sRNA_LacZ insert (C=48ng/uL) with XbaI and PstI

10 H2O

15 sRNA_LacZ insert

3 Buffer

1 PstI

1 XbaI

30uL Tot

37°C

20min

Digestion of sRNA_LacZ insert (C=45ng/uL) with SpeI and EcorI

10 H2O

15 sRNA_LacZ insert

3 Buffer

1 EcorI

1 SpeI

30uL Tot

37°C

20min

Purification on column

ligation:

Ligation sRNA_LacZ insert purif + pUC18

Digestion vector : 4,3 ng/ul

Digestion insert : 18

masse insert = 10*362/2175*50 = 83 ng

Vector : 11,6 uL

Insert : 4,6 uL

Buffer : 3 uL

T4 DNA ligase : 1,5 uL

H2O : 11 uL

Let incubate at 22°C for 30 min

Ligation sRNA_LacZ insert purif + litmus

Digestion vector : 13,3 ng/ul

Digestion insert : 11,4

masse insert = 10*376/3626*50 = 52 ng

Vector : 3.8 uL

Insert : 4,56 uL

Buffer : 2 uL

T4 DNA ligase : 1 uL

H2O : 8,64 uL

Let incubate at 22°C for 30 min

Preparing experiments of LacZ silencing

Plating MGZ1, F+ ; MG1655, F+ ; NEB Turbo on IPTG+Xgal