Paris/August 13

From 2007.igem.org

(Difference between revisions)
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== Digestions ==
== Digestions ==
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* L1.2 (AraC-pBAD BV) Spe Pst
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{|
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* L6.1 (rbs-CRE) Eco Pst
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|- style="background: #ccccff;"
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* L6.2 (rbs-CRE) Xba Pst
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! colspan="5" style="background: #ccccff;" | Digestion Products
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* L9 (lox66 FV) Eco Xba
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|- style="background: #ccccff; text-align: center;"
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* L10 (lox71 BV) Spe Pst
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|width=5%| Number
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* MP8.1 (gfp tri-part FI) Eco Spe
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|width=20%| Product Name
 +
|width=20%| Matrix Name
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|width=5%| Enzyme 1
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|width=5%| Enzyme 2
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|width=5%| Size
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|width=30%| Description
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|width=5%| Product Left
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|width=5%| []ng/µL
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|width=5%| Date
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|- style="background: #cccccc;" 
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| style="background: #FF99FF;" |D41
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|araC/pBad-J61002 plasmid BV
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|L1.2 (araC/pBad-J61002 plasmid)
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|Spe1
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|Pst1
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|
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|Vector with the pBAD promoter ready for insertion of a RBS/CDS insert
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|
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|
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|[[Paris/July 13| July 13]]
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D22
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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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|
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|
 +
|
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|
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|[[Paris/July 13| July 13]]
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D22
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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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|
 +
|
 +
|
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|
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|[[Paris/July 13| July 13]]
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D33
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|RBS(B0030)-CreORF BI
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|L6.2 (rbs-CRE)
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|XbaI
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|PstI
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|
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|RBS-Cre digestion for use as a BI
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|
 +
|
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|[[Paris/July 13| July 13]]
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D42
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|Lox66 FV
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|L9
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|EcoRI
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|XbaI
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|
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|pSB1A2-lox66 as a Forward vector
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|
 +
|
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|[[Paris/July 13| July 13]]
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D35
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|Lox71 BV
 +
|L10
 +
|SpeI
 +
|PstI
 +
|
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|pSB1A2-lox71 as a Backward vector
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|
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|
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|[[Paris/July 13| July 13]]
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D43
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|gfp-tripart FI
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|MP8.1
 +
|EcoRI
 +
|SpeI
 +
|
 +
|gfp-tri part (strongRBS-ORF-T) ready for use as a forward insert
 +
|
 +
|
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|[[Paris/July 13| July 13]]
 +
|}
digestion mix:
digestion mix:

Revision as of 15:43, 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 July 13
D22 pSB1A2 L6.1 (rbs-CRE) EcoR1 Pst1 July 13
D22 pSB1A2 L6.1 (rbs-CRE) EcoR1 Pst1 July 13
D33 RBS(B0030)-CreORF BI L6.2 (rbs-CRE) XbaI PstI RBS-Cre digestion for use as a BI July 13
D42 Lox66 FV L9 EcoRI XbaI pSB1A2-lox66 as a Forward vector July 13
D35 Lox71 BV L10 SpeI PstI pSB1A2-lox71 as a Backward vector July 13
D43 gfp-tripart FI MP8.1 EcoRI SpeI gfp-tri part (strongRBS-ORF-T) ready for use as a forward insert July 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
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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