Paris/August 13

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== Digestions ==
== Digestions ==
-
* L1.2 (AraC-pBAD BV) Spe Pst
+
{|
-
* L6.1 (rbs-CRE) Eco Pst
+
|- style="background: #ccccff;"
-
* L6.2 (rbs-CRE) Xba Pst
+
! colspan="5" style="background: #ccccff;" | Digestion Products
-
* L9 (lox66 FV) Eco Xba
+
|- style="background: #ccccff; text-align: center;"
-
* L10 (lox71 BV) Spe Pst
+
|width=5%| Number
-
* MP8.1 (gfp tri-part FI) Eco Spe
+
|width=20%| Product Name
 +
|width=20%| Matrix Name
 +
|width=5%| Enzyme 1
 +
|width=5%| Enzyme 2
 +
|width=5%| Size
 +
|width=30%| Description
 +
|width=5%| Product Left
 +
|width=5%| []ng/µL
 +
|width=5%| Date
 +
|- style="background: #cccccc;" 
 +
| style="background: #FF99FF;" |D41
 +
|araC/pBad-J61002 plasmid BV
 +
|L1.2 (araC/pBad-J61002 plasmid)
 +
|Spe1
 +
|Pst1
 +
|
 +
|Vector with the pBAD promoter ready for insertion of a RBS/CDS insert
 +
|
 +
|
 +
|[[Paris/August 13| August 13]]
 +
|- style="background: #cccccc;" 
 +
| style="background: #FF99FF;" |D22
 +
|<bbpart>pSB1A2</bbpart>
 +
|L6.1 (rbs-CRE)
 +
|EcoR1
 +
|Pst1
 +
|
 +
|
 +
|
 +
|
 +
|[[Paris/August 13| August 13]]
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D33
 +
|RBS(B0030)-CreORF BI
 +
|L6.2 (rbs-CRE)
 +
|XbaI
 +
|PstI
 +
|
 +
|RBS-Cre digestion for use as a BI
 +
|
 +
|
 +
|[[Paris/August 13| August 13]]
 +
|- style="background: #cccccc;" 
 +
| style="background: #FF99FF;" |D42
 +
|Lox66 FV
 +
|L9
 +
|EcoRI
 +
|XbaI
 +
|
 +
|pSB1A2-lox66 as a Forward vector
 +
|
 +
|
 +
|[[Paris/August 13| August 13]]
 +
|- style="background: #cccccc;" 
 +
| style="background: #FF99FF;" |D35
 +
|Lox71 BV
 +
|L10
 +
|SpeI
 +
|PstI
 +
|
 +
|pSB1A2-lox71 as a Backward vector
 +
|
 +
|
 +
|[[Paris/August 13| August 13]]
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D43
 +
|gfp-tripart FI
 +
|MP8.1
 +
|EcoRI
 +
|SpeI
 +
|
 +
|gfp-tri part (strongRBS-ORF-T) ready for use as a forward insert
 +
|
 +
|
 +
|[[Paris/August 13| August 13]]
 +
|}
digestion mix:
digestion mix:
Line 16: Line 91:
* 2µl Enzyme1
* 2µl Enzyme1
* 2µl Enzyme2
* 2µl Enzyme2
-
 
== Ligations ==
== Ligations ==
Line 39: Line 113:
|2
|2
|control of ligation efficiency
|control of ligation efficiency
-
|
+
|~1000
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |C2
| style="background: #ccffcc;" |C2
Line 47: Line 121:
|2µl
|2µl
|control of D35 digestion
|control of D35 digestion
-
|
+
|5
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |C4
| style="background: #ccffcc;" |C4
|
|
|
|
-
|D? (L9 lox66 EX)
+
|D42 (L9 lox66 EX)
|2µl
|2µl
-
|control of D? digestion
+
|control of D42 digestion
-
|
+
|5
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |L26
| style="background: #ccffcc;" |L26
|D33.2 (L6.2 RBS-Cre XP)
|D33.2 (L6.2 RBS-Cre XP)
|5
|5
-
|D? (L1 J61002::pbad SP)
+
|D41 (L1 J61002::pbad SP)
|1
|1
| Insertion of RBS-Cre behind araC/pBad
| Insertion of RBS-Cre behind araC/pBad
-
|
+
|x100
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |L27
| style="background: #ccffcc;" |L27
|D27 (ftsZ PCR XP)
|D27 (ftsZ PCR XP)
|5
|5
-
|D? (L1 J61002::pbad SP)
+
|D41 (L1 J61002::pbad SP)
|1
|1
| Insertion of ftsZ behind araC/pBad  
| Insertion of ftsZ behind araC/pBad  
-
|
+
|6
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |L28
| style="background: #ccffcc;" |L28
|D32 (ftsAZ PCR XP)
|D32 (ftsAZ PCR XP)
|5
|5
-
|D? (L1 J61002::pbad SP)
+
|D41 (L1 J61002::pbad SP)
|1
|1
| Insertion of ftsAZ behind araC/pBad  
| Insertion of ftsAZ behind araC/pBad  
-
|
+
|8
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |L29
| style="background: #ccffcc;" |L29
-
|D? (MP8.1 gfp-tripart ES)
+
|D43 (MP8.1 gfp-tripart ES)
|5
|5
-
|D? (L9 pSB1A2::lox66 XP)
+
|D42 (L9 pSB1A2::lox66 EX)
|2
|2
| Insertion of gfp-tripart in front of lox66  
| Insertion of gfp-tripart in front of lox66  
-
|
+
|x100
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L30(.1 & .2)
+
| style="background: #ccffcc;" |L30(A&B)
|dapAp [O31+O32]0.2µM
|dapAp [O31+O32]0.2µM
|2
|2
-
|D9.1 & D9.2 (dapAp [O31+O32]0.2µM)
+
|D9.1 & D9.2  
|1
|1
|cloning dapAp promotor in J61002
|cloning dapAp promotor in J61002
-
|
+
|x100
|}
|}

Latest revision as of 23:21, 16 August 2007

yesterday -- tomorrow

Digestions

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description Product Left []ng/µL Date
D41 araC/pBad-J61002 plasmid BV L1.2 (araC/pBad-J61002 plasmid) Spe1 Pst1 Vector with the pBAD promoter ready for insertion of a RBS/CDS insert August 13
D22 pSB1A2 L6.1 (rbs-CRE) EcoR1 Pst1 August 13
D33 RBS(B0030)-CreORF BI L6.2 (rbs-CRE) XbaI PstI RBS-Cre digestion for use as a BI August 13
D42 Lox66 FV L9 EcoRI XbaI pSB1A2-lox66 as a Forward vector August 13
D35 Lox71 BV L10 SpeI PstI pSB1A2-lox71 as a Backward vector August 13
D43 gfp-tripart FI MP8.1 EcoRI SpeI gfp-tri part (strongRBS-ORF-T) ready for use as a forward insert August 13

digestion mix:

  • 25µl plasmid
  • 1µl BSA
  • 10 µl NEB2
  • 60µl H2O
  • 2µl Enzyme1
  • 2µl Enzyme2

Ligations

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
C1 MP3.1 digested with EcoRI (D0) 2 control of ligation efficiency ~1000
C2 D35 (L10 lox71 dig.SP) 2µl control of D35 digestion 5
C4 D42 (L9 lox66 EX) 2µl control of D42 digestion 5
L26 D33.2 (L6.2 RBS-Cre XP) 5 D41 (L1 J61002::pbad SP) 1 Insertion of RBS-Cre behind araC/pBad x100
L27 D27 (ftsZ PCR XP) 5 D41 (L1 J61002::pbad SP) 1 Insertion of ftsZ behind araC/pBad 6
L28 D32 (ftsAZ PCR XP) 5 D41 (L1 J61002::pbad SP) 1 Insertion of ftsAZ behind araC/pBad 8
L29 D43 (MP8.1 gfp-tripart ES) 5 D42 (L9 pSB1A2::lox66 EX) 2 Insertion of gfp-tripart in front of lox66 x100
L30(A&B) dapAp [O31+O32]0.2µM 2 D9.1 & D9.2 1 cloning dapAp promotor in J61002 x100

Ligation mix:

  • 2µl 10x buffer
  • 1µl Ligase
  • XµL plasmid DNA solution
  • YµL insert DNA solution
  • H2O qsp 20µL

Reaction in 20µl

ON @ 4°C

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