Paris/August 13

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< Paris(Difference between revisions)
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|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |D22
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| style="background: #FF99FF;" |D22
|<bbpart>pSB1A2</bbpart>
|<bbpart>pSB1A2</bbpart>
|L6.1 (rbs-CRE)
|L6.1 (rbs-CRE)
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|<bbpart>pSB1A2</bbpart>
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|L6.1 (rbs-CRE)
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|EcoR1
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|Pst1
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| style="background: #ccffcc;" |D33
| style="background: #ccffcc;" |D33
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| style="background: #ccffcc;" |D42
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|Lox66 FV
|Lox66 FV
|L9
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|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |D35
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|Lox71 BV
|Lox71 BV
|L10
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|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |D43
| style="background: #ccffcc;" |D43
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* 2µl Enzyme1
* 2µl Enzyme1
* 2µl Enzyme2
* 2µl Enzyme2
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== Ligations ==
== Ligations ==
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|2
|2
|control of ligation efficiency
|control of ligation efficiency
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|~1000
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |C2
| style="background: #ccffcc;" |C2
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|2µl
|2µl
|control of D35 digestion
|control of D35 digestion
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|5
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |C4
| style="background: #ccffcc;" |C4
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|D? (L9 lox66 EX)
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|D42 (L9 lox66 EX)
|2µl
|2µl
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|control of D? digestion
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|control of D42 digestion
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|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
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|D? (L1 J61002::pbad SP)
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|D41 (L1 J61002::pbad SP)
|1
|1
| Insertion of RBS-Cre behind araC/pBad
| Insertion of RBS-Cre behind araC/pBad
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|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
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|D? (L1 J61002::pbad SP)
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|D41 (L1 J61002::pbad SP)
|1
|1
| Insertion of ftsZ behind araC/pBad  
| Insertion of ftsZ behind araC/pBad  
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|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
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|D? (L1 J61002::pbad SP)
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|D41 (L1 J61002::pbad SP)
|1
|1
| Insertion of ftsAZ behind araC/pBad  
| Insertion of ftsAZ behind araC/pBad  
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|8
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |L29
| style="background: #ccffcc;" |L29
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|D? (MP8.1 gfp-tripart ES)
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|D43 (MP8.1 gfp-tripart ES)
|5
|5
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|D? (L9 pSB1A2::lox66 XP)
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|D42 (L9 pSB1A2::lox66 EX)
|2
|2
| Insertion of gfp-tripart in front of lox66  
| Insertion of gfp-tripart in front of lox66  
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|x100
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |L30(.1 & .2)
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| style="background: #ccffcc;" |L30(A&B)
|dapAp [O31+O32]0.2µM
|dapAp [O31+O32]0.2µM
|2
|2
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|D9.1 & D9.2 (dapAp [O31+O32]0.2µM)
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|D9.1 & D9.2  
|1
|1
|cloning dapAp promotor in J61002
|cloning dapAp promotor in J61002
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|x100
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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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