Paris/August 17

From 2007.igem.org

(Difference between revisions)
Line 9: Line 9:
*L30A1 & L30B1 (dapAp>>mRFP in J61002)
*L30A1 & L30B1 (dapAp>>mRFP in J61002)
*MP1.1 (pJ23100) & MP2.1 (pJ23107)
*MP1.1 (pJ23100) & MP2.1 (pJ23107)
 +
 +
 +
 +
== Ligations ==
 +
 +
The following ligation reactions have been performed:
 +
 +
{|
 +
|- 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;" |C1
 +
|
 +
|
 +
|MP3.1 digested with EcoRI (D0)
 +
|2
 +
|control of ligation efficiency
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C3
 +
|
 +
|
 +
|D34 (lox66 BV)
 +
|4µl
 +
|control of D34 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C5
 +
|
 +
|
 +
|D50 (<bbpart>BBa_J61002</bbpart> BV)
 +
|1.5µl
 +
|control of D50 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C6
 +
|
 +
|
 +
|D41 (araC/pBad-J61002 plasmid SP dig.)
 +
|1µl
 +
|control of D41 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C7
 +
|
 +
|
 +
|D22 (pSB1A2 open vector EP)
 +
|4µl
 +
|control of D22 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C8
 +
|
 +
|
 +
|D47 (<bbpart>pSB1A2</bbpart> XP dig.)
 +
|4µl
 +
|control of D47 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C9
 +
|
 +
|
 +
|D48 (<bbpart>pSB1A2</bbpart> ES dig.)
 +
|2µl
 +
|control of D48 digestion
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L31
 +
|D44 (<bbpart>B0015</bbpart> terminator BI)
 +
|4
 +
|D35 (lox71 BV)
 +
|3
 +
| Insertion of B0015 terminator behind lox71
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L32
 +
|D45 (<bbpart>B0015</bbpart> terminator FI)
 +
|4
 +
|D42 (Lox66 FV)
 +
|3
 +
| Insertion of B0015 terminator in front of lox66
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L33
 +
|D33.1 (RBS(B0030)CreORF BI)
 +
|5
 +
|D50 (<bbpart>BBa_J61002</bbpart> BV)
 +
|1.5
 +
| pTet>>RBS-Cre
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L34
 +
|D49 (lox71-gfptripart BI)
 +
|5
 +
|D50 (<bbpart>BBa_J61002</bbpart> BV)
 +
|1.5
 +
|pTet>>lox71-gfptripart
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L35
 +
|D49 (lox71-gfptripart BI)
 +
|5
 +
|D41 (araC/pBad-J61002 plasmid SP dig.)
 +
|1
 +
|araC/pBad>>lox71-gfptripart
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L36
 +
|D13.4 (<bbpart>mRFP</bbpart> dig. SP)
 +
|6
 +
|D34 (lox66 BV)
 +
|4
 +
|lox66-mRFP
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L37
 +
|D52 (RBS-dapAcoli BI)
 +
|6
 +
|D34 (lox66 BV)
 +
|4
 +
|lox66-RBS-dapAcoli BI
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L38
 +
|D54 (RBS-dapAsubtilis BI)
 +
|13
 +
|D34 (lox66 BV)
 +
|4
 +
|lox66-RBS-dapAsubtilis BI
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L39
 +
|D51 (RBS-dapAcoli BBinsert)
 +
|4
 +
|D22 (pSB1A2 open vector EP)
 +
|4µl
 +
|RBS-dapAcoli cloned in pSB1A2
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L40
 +
|D53 (RBS-dapAsubtilis BBinsert)
 +
|13 (
 +
|D22 (pSB1A2 open vector EP)
 +
|4µl
 +
|RBS-dapAsubtilis cloned in pSB1A2
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L41
 +
|D27' (lox71-ftsZ bw-insert)
 +
|12
 +
|D47 (<bbpart>pSB1A2</bbpart> XP dig.)
 +
|4µl
 +
|lox71-ftsZ cloned in pSB1A2
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L42
 +
|D32 (lox71-ftsAftsZ bw-insert)
 +
|6
 +
|D47 (<bbpart>pSB1A2</bbpart> XP dig.)
 +
|4µl
 +
|lox71-ftsA-Z cloned in pSB1A2
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L43
 +
|D55 (RBS-dapAsubtilis FI)
 +
|15
 +
|D48 (<bbpart>pSB1A2</bbpart> ES dig.)
 +
|2µl
 +
|RBS-dapAsubtilis cloned in pSB1A2
 +
|
 +
|}
 +
 +
Ligation mix:
 +
*2µl 10x buffer
 +
*1µl Ligase
 +
*XµL plasmid DNA solution
 +
*YµL insert DNA solution
 +
*H2O qsp 20µL
 +
Reaction in 20µl
 +
 +
ON @ 4°C

Revision as of 17:03, 18 August 2007

Miniprep rxn

The following minipreps were performed:

  • L26.1 & L26.2 (araC/pbad>>RBS-Cre)
  • L27.2, L27.3, L27.4 & L27.5 (araC/pbad>>ftsZ)
  • L28.2, L28.3 & L28.8 (araC/pBad>>ftsA-ftsZ)
  • L29.1 & L29.2 (lox66-gfptripart)
  • L30A1 & L30B1 (dapAp>>mRFP in J61002)
  • MP1.1 (pJ23100) & MP2.1 (pJ23107)


Ligations

The following ligation reactions have been performed:

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
C3 D34 (lox66 BV) 4µl control of D34 digestion
C5 D50 (BBa_J61002 BV) 1.5µl control of D50 digestion
C6 D41 (araC/pBad-J61002 plasmid SP dig.) 1µl control of D41 digestion
C7 D22 (pSB1A2 open vector EP) 4µl control of D22 digestion
C8 D47 (pSB1A2 XP dig.) 4µl control of D47 digestion
C9 D48 (pSB1A2 ES dig.) 2µl control of D48 digestion
L31 D44 (B0015 terminator BI) 4 D35 (lox71 BV) 3 Insertion of B0015 terminator behind lox71
L32 D45 (B0015 terminator FI) 4 D42 (Lox66 FV) 3 Insertion of B0015 terminator in front of lox66
L33 D33.1 (RBS(B0030)CreORF BI) 5 D50 (BBa_J61002 BV) 1.5 pTet>>RBS-Cre
L34 D49 (lox71-gfptripart BI) 5 D50 (BBa_J61002 BV) 1.5 pTet>>lox71-gfptripart
L35 D49 (lox71-gfptripart BI) 5 D41 (araC/pBad-J61002 plasmid SP dig.) 1 araC/pBad>>lox71-gfptripart
L36 D13.4 (mRFP dig. SP) 6 D34 (lox66 BV) 4 lox66-mRFP
L37 D52 (RBS-dapAcoli BI) 6 D34 (lox66 BV) 4 lox66-RBS-dapAcoli BI
L38 D54 (RBS-dapAsubtilis BI) 13 D34 (lox66 BV) 4 lox66-RBS-dapAsubtilis BI
L39 D51 (RBS-dapAcoli BBinsert) 4 D22 (pSB1A2 open vector EP) 4µl RBS-dapAcoli cloned in pSB1A2
L40 D53 (RBS-dapAsubtilis BBinsert) 13 ( D22 (pSB1A2 open vector EP) 4µl RBS-dapAsubtilis cloned in pSB1A2
L41 D27' (lox71-ftsZ bw-insert) 12 D47 (pSB1A2 XP dig.) 4µl lox71-ftsZ cloned in pSB1A2
L42 D32 (lox71-ftsAftsZ bw-insert) 6 D47 (pSB1A2 XP dig.) 4µl lox71-ftsA-Z cloned in pSB1A2
L43 D55 (RBS-dapAsubtilis FI) 15 D48 (pSB1A2 ES dig.) 2µl RBS-dapAsubtilis cloned in pSB1A2

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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