Team:Paris Bettencourt/Notebook/Trojan Horse/Tuesday 20th August.html

Trojan Horse

ASDF

20^th August

CLONING the target Plasmids (Kan and lacZ)

Gel purification of LacZ using Thermo Scientific Kit

lacZ : 83 ng/ul

Gel purification of Kan using Thermo Scientific Kit

Kan : 80ng/ul

Digestion of pACYC (pT002) with EcoRI and XhoI

10 H2O

2 Buffer

4 pACYC

2 EcoRI

2 XhoI

20uL Tot

37°C

20min

Digestion of pACYC (pT002) with BglII and NcoI

10 H2O

2 Buffer

4 pACYC

2 BglII

2 NcoI

20uL Tot

37°C

20min

Digestion of Kan insert with EcorI and XhoI

10 H2O

15 Kan insert

3 Buffer

1 EcoRI

1 XhoI

30uL Tot

37°C

20min

Digestion of LacZ insert with BglII and NcoI

10 H2O

15 LacZ insert

3 Buffer

1 BglII

1 NcoI

30uL Tot

37°C

20min

Gel of digestions

100Bp+ / pACYC+EcoRI+XhoI / pACYC+EcoRI+XhoI / pACYC+BglII+NcoI /pACYC+BglII+NcoI

Colum purification of Kan insert digestions using Thermo Scientific Kit

Column purification of LacZ insert digestions using Thermo Scientific Kit

Colum purification of pACYC digestions using Thermo Scientific Kit

Heat inactivation of Kan digestion

65°C

20min

Ligation Kan insert purif + pACYC (digestion EcoRI + XhoI)

Digestion vector : 6.8 ng/ul

Digestion insert : 14

masse insert = 10*1052/3770*50 = 140 ng

Vector : 7.35 uL

Insert : 10 uL

Buffer : 2 uL

T4 DNA ligase : 0,2 uL

Let incubate at 22°C for 30 min

Ligation Kan insert heat desactivated + pACYC (digestion EcoRI + XhoI)

Digestion vector : 6.8 ng/ul

Digestion insert : 40/2 = 20

masse insert = 10*1052/3770*50 = 140 ng

Vector : 7.35 uL

Insert : 7 uL

Buffer : 2 uL

T4 DNA ligase : 0,2 uL

H2O : 4 uL

Let incubate at 22°C for 30 min

Ligation LacZ insert purif + pACYC (pigestion BglII + NcoI)

Digestion vector : 10? ng/ul

Digestion insert : 33

masse insert = 10*449/3770*50 = 59 ng

Vector : 5 uL

Insert : 1.8 uL

Buffer : 2 uL

T4 DNA ligase : 0,2 uL

H2O : 11 uL

Let incubate at 22°C for 30 min

Chemical transformation in NEB turbo

Reference protocol

Recovery 1 hour.

Plate on

Tube 1 = negative control

Tube 2 : Kan (purif) Kan / Cm

Tube 3 : Kan (heat inactivated) Kan/ Cm

Tube 4 : lac(purify, enzyme cannot be heat inactivated) Cm/ IPTG/ Xgal

Cloning the Weapon (ie sRNA into Litmus and pUC 18)

Gel of litmus PCR

Gel purification of litmus PCR using Thermo Scientific Kit

PCR from mini Gene sRNA anti Kan

35 H2O

10 Buffer 5x

1 dNTP

2 FW iT009

2 RV iT0010

0.2 minigene-plasmid amp from IDT

0.5 phusion

PCR Phusion Gradient around 53 °C

Time : 30 secondes

Gibson Assembly

Protocol from the Kit, quantities are divided by 2: final volume of reaction = 10uL

Assembly of pUC18-SRNA_KAN : 5uL MasterMix, 0.5uL H2O, 3uL pUC18 with overhang from PCR (nanodrop = 13,4), 1,5uL sRNA from PCR of minigene (nanodrop = 26,7)

Assembly of Litmus28i-GFP-SRNA_KAN : 5uL MasterMix, 3,6uL H2O, 0,5uL Litmus28i-GFP with overhang from PCR (nanodrop = 85), 0,9uL sRNA from PCR of minigene (nanodrop = 26,7)

Heat shock Transformation into provided competent cells. 60 min recovery, plate on AMP.

Regular cloning using biobricks

Digestion

Insert sRNA cut by XbaI & PstI

Vector Litmus cut by PstI & SpeI

Insert sRNA cut by EcoRI & SpeI

Vector Litmus cut by EcoRI & SpeI

Digestion of Vectors

10 H2O

2 Buffer

4 Vector

2 Enz 1

2 Enz 2

20uL Tot

37°C

20min

Digestion of Inserts

10 H2O

15 insert

3 Buffer

1 Enz 1

1 Enz 2

30uL Tot

37°C

20min

Heat inactivation before ligation

80°C for 20 minutes

Ligation

pUC= 40ng in 20 uL => 3ng/ul

litmus 340ng =>17ng /ul

insert 420 ng => 14 ng / uL

Litmus :

7uL vector

8 ul insert

2 uL Buffer

O,2 uLT4 DNA ligase

3ul Water

Vtot = 20 uL

pUC

20 uL vector

9 uL insert

4 uL Buffer

0,4 T4 DNA

7 uL water

Vtot = 40uL

Chemical transformation in NEB turbo

Tube 1 : negative control

Tube 2 lig litmus

Tube 3 lig pUC

• plate everything on amp