Paris/August 13

From 2007.igem.org

(Difference between revisions)
(→Digestions)
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* 2µl Enzyme1
* 2µl Enzyme1
* 2µl Enzyme2
* 2µl Enzyme2
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 +
 +
== Ligations ==
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 +
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{|
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|- style="background: #ccccff;"
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! colspan="5" style="background: #ccccff;" | Ligations
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|- style="background: #ccccff; text-align: center;"
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|width=5%| Number
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|width=20%| Insert
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|width=5%| Insert Volume (µL)
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|width=25%| Vector
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|width=5%| Vector Volume (µL)
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|width=35%| Comments
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|width=5%| Number of colonies
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C1
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|
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|
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|MP3.1 digested with EcoRI (D0)
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|2
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|control of ligation efficiency
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C2
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|
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|
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|D35 (L10 lox71 dig.SP)
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|2µl
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|control of D35 digestion
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C4
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|
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|
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|D? (L9 lox66 EX)
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|2µl
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|control of D? digestion
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L26
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|D33.2 (L6.2 RBS-Cre XP)
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|5
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|D? (L1 J61002::pbad SP)
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|1
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| Insertion of RBS-Cre behind araC/pBad
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L27
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|D27 (ftsZ PCR XP)
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|5
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|D? (L1 J61002::pbad SP)
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|1
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| Insertion of ftsZ behind araC/pBad
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L28
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|D32 (ftsAZ PCR XP)
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|5
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|D? (L1 J61002::pbad SP)
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|1
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| Insertion of ftsAZ behind araC/pBad
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L29
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|D? (MP8.1 gfp-tripart ES)
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|5
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|D? (L9 pSB1A2::lox66 XP)
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|2
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| Insertion of gfp-tripart in front of lox66
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L30(.1 & .2)
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|dapAp [O31+O32]0.2µM
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|2
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|D9.1 & D9.2 (dapAp [O31+O32]0.2µM)
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|1
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|cloning dapAp promotor in J61002
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|
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|}
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Ligation mix:
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*2µl 10x buffer
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*1µl Ligase
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*XµL plasmid DNA solution
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*YµL insert DNA solution
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*H2O qsp 20µL
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Reaction in 20µl
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 +
ON @ 4°C

Revision as of 11:27, 14 August 2007

Digestions

  • L1.2 (AraC-pBAD BV) Spe Pst
  • L6.1 (rbs-CRE) Eco Pst
  • L6.2 (rbs-CRE) Xba Pst
  • L9 (lox66 FV) Eco Xba
  • L10 (lox71 BV) Spe Pst
  • MP8.1 (gfp tri-part FI) Eco Spe

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
C2 D35 (L10 lox71 dig.SP) 2µl control of D35 digestion
C4 D? (L9 lox66 EX) 2µl control of D? digestion
L26 D33.2 (L6.2 RBS-Cre XP) 5 D? (L1 J61002::pbad SP) 1 Insertion of RBS-Cre behind araC/pBad
L27 D27 (ftsZ PCR XP) 5 D? (L1 J61002::pbad SP) 1 Insertion of ftsZ behind araC/pBad
L28 D32 (ftsAZ PCR XP) 5 D? (L1 J61002::pbad SP) 1 Insertion of ftsAZ behind araC/pBad
L29 D? (MP8.1 gfp-tripart ES) 5 D? (L9 pSB1A2::lox66 XP) 2 Insertion of gfp-tripart in front of lox66
L30(.1 & .2) dapAp [O31+O32]0.2µM 2 D9.1 & D9.2 (dapAp [O31+O32]0.2µM) 1 cloning dapAp promotor in J61002

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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