Team:Paris Bettencourt/Notebook/Trojan Horse/Monday 26th August.html

Trojan Horse

ASDF

26^th August

Place your twit here

Digestion of litmus miniprep (C=280ng/uL)with PstI and SpeI

13 H2O

3 litmus miniprep

2 Buffer

1 PstI

1 SpeI

20uL Tot

37°C

20min

Digestion of sRNA_Cm insert (C=31ng/uL) with XbaI and PstI

10 H2O

15 sRNA_Cm insert

3 Buffer

1 XbaI

1 PstI

30uL Tot

37°C

20min

PCR purification of litmus digestion using Thermo Scientific Kit

PCR purification of sRNA_Cm insert digestion using Thermo Scientific Kit

Ligation sRNA_Cm insert purif + litmus

Digestion vector : 13,3 ng/ul

Digestion insert : 10

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

Vector : 3.8 uL

Insert : 5.2 uL

Buffer : 2 uL

T4 DNA ligase : 1 uL

H2O : 8 uL

Let incubate at 22°C for 30 min

Ligation control litmus

Vector : 3.8 uL

Insert : 0 uL

Buffer : 2 uL

T4 DNA ligase : 1 uL

H2O : 13,2 uL

Let incubate at 22°C for 30 min

Digestion of pUC18 PCR (C=13ng/uL) with EcorI and SpeI

10 H2O

15 pUC18 PCR

3 Buffer

1 EcorI

1 SpeI

30uL Tot

37°C

20min

Digestion of sRNA_Cm insert (C=31ng/uL) with SpeI and EcorI

10 H2O

15 sRNA_Cm insert

3 Buffer

1 EcorI

1 SpeI

30uL Tot

37°C

20min

PCR purification of pUC18 digestion using Thermo Scientific Kit

PCR purification of sRNA_Cm insert digestion using Thermo Scientific Kit

Ligation sRNA_Cm insert purif + pUC18

Digestion vector : 4,3 ng/ul

Digestion insert : 9,4

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

Vector : 11,6 uL

Insert : 8,8 uL

Buffer : 3 uL

T4 DNA ligase : 1,5 uL

H2O : 7,1 uL

Let incubate at 22°C for 30 min

Ligation control pUC18

Vector : 11,6 uL

Insert : 0 uL

Buffer : 3 uL

T4 DNA ligase : 1,5 uL

H2O : 15,9 uL

Let incubate at 22°C for 30 min

Chemical transformation in NEB-Turbo only (control -)

Chemical transformation in NEB-Turbo of ligation sRNA_Cm insert purif + litmus

Chemical transformation in NEB-Turbo of ligation control litmus

Chemical transformation in NEB-Turbo of ligation sRNA_Cm insert purif + pUC18

Chemical transformation in NEB-Turbo of ligation control pUC18

Chemical transformation in NEB-Turbo of litmus (control +)

Plate all 5 on Amp

PCR from mini Gene sRNA anti LacZ

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

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