Paris/October 20

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< Paris(Difference between revisions)
(→Digestion Reaction)
 
(3 intermediate revisions not shown)
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[[Paris/October 19|yesterday]] -- [[Paris/October 21|tomorrow]] <br>
[[Paris/October 19|yesterday]] -- [[Paris/October 21|tomorrow]] <br>
== Digestion Reaction ==
== Digestion Reaction ==
 +
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Digestion Products
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=24%| Product Name
 +
|width=24%| Matrix Name
 +
|width=5%| Enzyme 1
 +
|width=5%| Enzyme 2
 +
|width=5%| Size
 +
|width=34%| Description
 +
|width=1%|
 +
|width=1%|
 +
|width=1%|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D79
 +
|pJ23100>>lox71-B0015(T)
 +
|L44.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D80
 +
|pJ23100>>lox71-B0015(T)
 +
|L44.2
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D81
 +
|pJ23107>>lox71-B0015(T)
 +
|L45.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D82
 +
|pJ23107>>lox71-B0015(T)
 +
|L45.2
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D83
 +
|pTet>>lox71-B0015(T)
 +
|L46.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D84
 +
|araC/pBad>>lox71-B0015(T)
 +
|L47.1
 +
|Xba1
 +
|Pst1
 +
|
 +
|BI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D85
 +
|B0015(T)-lox66-RBSDapAcoli
 +
|L53.7
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D86
 +
|attB-lox66-RBSDapAcoli
 +
|L54.3
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D87
 +
|attB-lox66-RBSDapAcoli
 +
|L54.4
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D88
 +
|B0015(T)-lox66-RBSdapAsubtilis
 +
|L56.3
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D89
 +
|attB-lox66-RBSdapAsubtilis
 +
|L57.1
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D90
 +
|attB-lox66-RBSdapAsubtilis
 +
|L57.3
 +
|EcoRI
 +
|SpeI
 +
|
 +
|FI
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D91
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.1
 +
|EcoRI
 +
|XbaI
 +
|
 +
|FV - Medium constitutive promoter in J61002 (cl. 1) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D92
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.2
 +
|EcoRI
 +
|XbaI
 +
|
 +
|FV - Medium constitutive promoter in J61002 (cl. 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D93
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.1
 +
|Spe1
 +
|Pst1
 +
|
 +
|BV - Medium constitutive promoter in J61002 (cl. 1) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D94
 +
|<bbpart>BBa_J61025</bbpart>
 +
|MP14.2
 +
|Spe1
 +
|Pst1
 +
|
 +
|BV - Medium constitutive promoter in J61002 (cl. 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
 +
|
 +
|
 +
|
 +
|}
== PCR reaction ==
== PCR reaction ==
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL
 +
|MgCl2= 10µM 0µL (no extra)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O55
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=YES
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL  ! GC one !
 +
|MgCl2= 10µM 0µL (no extra)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O55
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=NO
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL ! GC one !
 +
|MgCl2= 10µM 0µL (no extra)
 +
|DMSO= 2,5µL (or 5%)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O55
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=NO
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-T1T2-ufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL
 +
|MgCl2= 10µM 0µL (no extra)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O56
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=YES
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL  ! GC one !
 +
|MgCl2= 10µM 0µL (no extra)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O56
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=NO
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL ! GC one !
 +
|MgCl2= 10µM 0µL (no extra)
 +
|DMSO= 2,5µL (or 5%)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O56
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=YES
 +
|Image=
 +
|Band=
 +
|}}
== Purification of Digestion and PCR products ==
== Purification of Digestion and PCR products ==
== Ligation Reactions ==
== Ligation Reactions ==
 +
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Ligations
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=20%| Insert
 +
|width=5%| Insert Volume (µL)
 +
|width=25%| Vector
 +
|width=5%| Vector Volume (µL)
 +
|width=35%| Comments
 +
|width=5%| Number of colonies
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C22
 +
|
 +
|
 +
|D47
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C23
 +
|
 +
|
 +
|D72
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C24
 +
|
 +
|
 +
|D72 (from Oct. 14)
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C25
 +
|
 +
|
 +
|D50
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C26
 +
|
 +
|
 +
|D41
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C27
 +
|
 +
|
 +
|D22
 +
|5
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C28
 +
|
 +
|
 +
|D81
 +
|11
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C29
 +
|
 +
|
 +
|D89
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C30
 +
|
 +
|
 +
|D90
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L68
 +
|D56 - araC/pBad>>RBS-Cre BBinsert
 +
|10
 +
|D72 - pSB2K3 BBvector
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L68 bis
 +
|D56 (14/10/07) - araC/pBad>>RBS-Cre BBinsert
 +
|10
 +
|D72 (14/10/07) - pSB2K3 BBvector
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L69
 +
|D92
 +
|10
 +
|D50
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L70
 +
|D92
 +
|10
 +
|D41
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L71
 +
|D93
 +
|15
 +
|D50
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L72
 +
|D93
 +
|15
 +
|D41
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L73
 +
|D84
 +
|10
 +
|D22
 +
|5
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L74
 +
|D80
 +
|3
 +
|D81
 +
|15
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L74 bis
 +
|D80
 +
|4
 +
|D81
 +
|12
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L75
 +
|D83
 +
|4
 +
|D89
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L76
 +
|D83
 +
|10
 +
|D90
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L77
 +
|D94.6
 +
|3
 +
|D95
 +
|10
 +
|
 +
|0
 +
|}
== Culture ==
== Culture ==
Launching of 80mL culture of the strain countaining the plasmid pY-2P-IntC, last minipreps revealed the weak nomber of plasmid recover with a classic miniprep on 5mL culture.
Launching of 80mL culture of the strain countaining the plasmid pY-2P-IntC, last minipreps revealed the weak nomber of plasmid recover with a classic miniprep on 5mL culture.

Latest revision as of 16:51, 23 October 2007

yesterday -- tomorrow

Contents

Digestion Reaction

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D79 pJ23100>>lox71-B0015(T) L44.1 Xba1 Pst1 BI
D80 pJ23100>>lox71-B0015(T) L44.2 Xba1 Pst1 BI
D81 pJ23107>>lox71-B0015(T) L45.1 Xba1 Pst1 BI
D82 pJ23107>>lox71-B0015(T) L45.2 Xba1 Pst1 BI
D83 pTet>>lox71-B0015(T) L46.1 Xba1 Pst1 BI
D84 araC/pBad>>lox71-B0015(T) L47.1 Xba1 Pst1 BI
D85 B0015(T)-lox66-RBSDapAcoli L53.7 EcoRI SpeI FI
D86 attB-lox66-RBSDapAcoli L54.3 EcoRI SpeI FI
D87 attB-lox66-RBSDapAcoli L54.4 EcoRI SpeI FI
D88 B0015(T)-lox66-RBSdapAsubtilis L56.3 EcoRI SpeI FI
D89 attB-lox66-RBSdapAsubtilis L57.1 EcoRI SpeI FI
D90 attB-lox66-RBSdapAsubtilis L57.3 EcoRI SpeI FI
D91 BBa_J61025 MP14.1 EcoRI XbaI FV - Medium constitutive promoter in J61002 (cl. 1)
D92 BBa_J61025 MP14.2 EcoRI XbaI FV - Medium constitutive promoter in J61002 (cl. 2)
D93 BBa_J61025 MP14.1 Spe1 Pst1 BV - Medium constitutive promoter in J61002 (cl. 1)
D94 BBa_J61025 MP14.2 Spe1 Pst1 BV - Medium constitutive promoter in J61002 (cl. 2)

PCR reaction

PCR : PrefixBB-frt1-CmR-frt2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL YES
72°C 1'40" Oligo R 10µM O55 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL  ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL NO
72°C 1'40" Oligo R 10µM O55 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL NO
72°C 1'40" Oligo R 10µM O55 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-T1T2-ufixBB
PCR Settings Buffer (5x) (5x) 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL YES
72°C 1'40" Oligo R 10µM O56 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL  ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL NO
72°C 1'40" Oligo R 10µM O56 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL YES
72°C 1'40" Oligo R 10µM O56 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL

Purification of Digestion and PCR products

Ligation Reactions

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
C22 D47 3 0
C23 D72 3 0
C24 D72 (from Oct. 14) 3 0
C25 D50 3 0
C26 D41 3 0
C27 D22 5 0
C28 D81 11 0
C29 D89 3 0
C30 D90 3 0
L68 D56 - araC/pBad>>RBS-Cre BBinsert 10 D72 - pSB2K3 BBvector 3 0
L68 bis D56 (14/10/07) - araC/pBad>>RBS-Cre BBinsert 10 D72 (14/10/07) - pSB2K3 BBvector 3 0
L69 D92 10 D50 3 0
L70 D92 10 D41 3 0
L71 D93 15 D50 3 0
L72 D93 15 D41 3 0
L73 D84 10 D22 5 0
L74 D80 3 D81 15 0
L74 bis D80 4 D81 12 0
L75 D83 4 D89 3 0
L76 D83 10 D90 3 0
L77 D94.6 3 D95 10 0

Culture

Launching of 80mL culture of the strain countaining the plasmid pY-2P-IntC, last minipreps revealed the weak nomber of plasmid recover with a classic miniprep on 5mL culture.

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