Team:Paris Bettencourt/Notebook/Trojan Horse/Monday 26th August.html
From 2013.igem.org
Trojan Horse
ASDF26th August
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