Paris/July 30

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== Ligations ==
== Ligations ==
-
Merci Eimad
+
 
 +
 
 +
{|
 +
|- 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;" |Control 1
 +
|
 +
|
 +
|MP3.1 digested with EcoRI (D0)
 +
|2
 +
|control of ligation efficiency
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |a faire
 +
|
 +
|
 +
|MP3.1 digested with EcoRI (D0)
 +
|2
 +
|without ligase in the mix: control of digestion efficiency
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |Control 2
 +
|
 +
|
 +
|D35
 +
|2µl
 +
|control of D35 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |Control 3
 +
|
 +
|
 +
|D34
 +
|2µl
 +
|control of D34 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L3''
 +
|D19 (lox66dapAsubtilis)
 +
|4
 +
|D22.1 (pSB1A2 dig. EcoRI&PstI)
 +
|2
 +
| Insertion of lox66DapASubtilis as a biobrick
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L5''
 +
|D21’(lox66DapASubtilis)
 +
|4
 +
|D1 (j23100 digested for CDS insertion)
 +
|2
 +
|construction of the biobrick J23100-lox66dapAsubtilis
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L7''
 +
|D27' (lox-ftsZ)
 +
|4
 +
|D1 (j23100 digested for CDS insertion)
 +
|2
 +
|construction of the biobrick J23100- lox-ftsZ
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L8''
 +
|D27' (lox-ftsZ)
 +
|4
 +
|D3 (j23107 digested for CDS insertion)
 +
|2
 +
|construction of the biobrick J23107- lox-ftsZ
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L9''
 +
|lox66 [O14+O15] (2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2
 +
|lox66 cloned as a biobrick in pSB1A2
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2
 +
|lox71 cloned as a biobrick in pSB1A2
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L11
 +
|D21’(lox66dapAsubtilis)
 +
|4
 +
|D3 (j23107 digested for CDS insertion)
 +
|2
 +
|construction of the biobrick: J23107 promotor-lox66dapAsubtilis
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L12
 +
|D26.1 (gfp-tripart)
 +
|2
 +
|D35 (lox66 plasmid digested as a BV)
 +
|4
 +
|construction of the biobrick lox66-gfp
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10''
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
 +
|D22.2 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
 +
|0
 +
|}
 +
 
 +
Ligation mix:
 +
*1µl 10x buffer
 +
*0.5µl Ligase
 +
*2µL plasmid DNA solution
 +
*4µL insert DNA solution
 +
 
 +
Reaction in 10µl
 +
 
 +
ON @ 4°C
==MiniPreps==
==MiniPreps==
* L1".1, L1".2  
* L1".1, L1".2  
* L6".1, L6".2
* L6".1, L6".2

Revision as of 11:00, 1 August 2007

Contents

Sequences

All the biobricks that we got from the plate and all the ligations that succeeded up to now.


Colonies PCR screen

On colony PCRs performed to screen for correct ligation reactions (performed on 27/07/07):

10 clones of L1 ligation reactions, 6 clones of L6 ligation reactions

preparation of the following PCR mix

  • 18x (12.5µL 2xreaction mix)= 225
  • 18x (0.5µL oligo O18 VF2 10µM)=9
  • 18x (0.5µL oligo O19 VR 10µM)=9
  • 12µL H20+colony (10min 100°)

PCRs

PCR : P5 lox66-DapAColi
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 985
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O6 2.5µL
2m00' Oligo R 10µM O7 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : P6 Lox66-DapASubtilis
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 979
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O8 2.5µL
2m00' Oligo R 10µM O9 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA 0.5µl Subtilis DNA


PCR : P7 Lox71-FtsZ
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 1260
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O1 2.5µL
2m00' Oligo R 10µM O2 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : P8 RBS-DapAColi
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 952
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O20 2.5µL
2m00' Oligo R 10µM O7 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : P11 Lox71-KmR-Lox66
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 1084
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O10 2.5µL
2m00' Oligo R 10µM O11 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA 0.5 µl MP12

Digestion reactions

The following digestion reactions have been performed:

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 size Description
D19' BB dig. lox66DapAsubtilis lox66-DapAsubtilis P6' PCR product EcoRI PstI ~960 PCR product digested for cloning as a BioBrick
D21' bw-insert lox66-DapAsubtilis lox66-DapAsubtilis P6' PCR product XbaI PstI ~960 PCR product digested for cloning as a BI
D27' lox71-ftsZ bw-insert lox71-ftsZ PCR product P7 XbaI PstI lox71-ftsZ(unmutated) ready for use as a backward insert
D33 RBS(B0030)CreORF L6' (clone 1 & 2) XbaI PstI RBS-Cre digestion for use as a BI
D34 lox66 L9' lox66 SpeI PstI Biobrick plasmid carrying lox66 digested as a BV
D35 lox71 L10' lox71 SpeI PstI Biobrick plasmid carrying lox71 digested as a BV

Ligations

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
Control 1 MP3.1 digested with EcoRI (D0) 2 control of ligation efficiency
a faire MP3.1 digested with EcoRI (D0) 2 without ligase in the mix: control of digestion efficiency
Control 2 D35 2µl control of D35 digestion
Control 3 D34 2µl control of D34 digestion
L3 D19 (lox66dapAsubtilis) 4 D22.1 (pSB1A2 dig. EcoRI&PstI) 2 Insertion of lox66DapASubtilis as a biobrick
L5 D21’(lox66DapASubtilis) 4 D1 (j23100 digested for CDS insertion) 2 construction of the biobrick J23100-lox66dapAsubtilis
L7 D27' (lox-ftsZ) 4 D1 (j23100 digested for CDS insertion) 2 construction of the biobrick J23100- lox-ftsZ
L8 D27' (lox-ftsZ) 4 D3 (j23107 digested for CDS insertion) 2 construction of the biobrick J23107- lox-ftsZ
L9 lox66 [O14+O15] (2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 lox66 cloned as a biobrick in pSB1A2
L10 lox71 [O16+O17] (2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 lox71 cloned as a biobrick in pSB1A2
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L11 D21’(lox66dapAsubtilis) 4 D3 (j23107 digested for CDS insertion) 2 construction of the biobrick: J23107 promotor-lox66dapAsubtilis
L12 D26.1 (gfp-tripart) 2 D35 (lox66 plasmid digested as a BV) 4 construction of the biobrick lox66-gfp
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0
L10 lox71 [O16+O17] (0.2µM) 2 D22.2 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick 0

Ligation mix:

  • 1µl 10x buffer
  • 0.5µl Ligase
  • 2µL plasmid DNA solution
  • 4µL insert DNA solution

Reaction in 10µl

ON @ 4°C

MiniPreps

  • L1".1, L1".2
  • L6".1, L6".2
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