Paris/September 18

From 2007.igem.org

(Difference between revisions)
(→DapA Wanner deletion: pKD13-DapA homologies PCR)
(→Ligation reactions)
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|width=5%| Number of colonies
|width=5%| Number of colonies
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |C1
+
| style="background: #ccffcc;" |C10
|
|
|
|
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|MP3.1 digested with EcoRI (D0)
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|gfptripart-lox66 BV <bbpart>pSB1A2</bbpart> (D69)
|2
|2
-
|control of ligation efficiency
+
|
 +
|
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|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C11
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|
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|
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|B0015(T)-lox66 BV (D58.1)
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|3
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|
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|
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|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C12
 +
|
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|
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|attB BV (D68)
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|2
 +
|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C13
 +
|
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|
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|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> BV (D64)
 +
|1.5
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C14
 +
|
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|
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|lox66-mRFP FV (D61.1)
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|1.5
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C15
 +
|
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|
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|<bbpart>B0015</bbpart> BV <bbpart>pSB1AK3</bbpart> (D46)
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|1
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|
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|
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|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C16
 +
|
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|
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|<bbpart>pSB2K3</bbpart> ES open vector (D73)
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|4
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C17
 +
|
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|
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|<bbpart>pSB2K3</bbpart> BBvector (D72)
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|2
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C18
 +
|
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|
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|pSB1A2 EP open vector (D22)
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|2
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L44
 +
|lox71-B0015(T) BI (D57.1)
 +
|15
 +
|<bbpart>pJ23100</bbpart> SP(D1)
 +
|2
 +
|pJ23100>>lox71-B0015(T)
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L45
 +
|lox71-B0015(T) BI (D57.1)
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|15
 +
|<bbpart>pJ23107</bbpart> SP(D3)
 +
|2
 +
|pJ23107>>lox71-B0015(T)
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L46
 +
|lox71-B0015(T) BI (D57.2)
 +
|15
 +
|<bbpart>BBa_J61002</bbpart> pTet SP dig.(D50)
 +
|2
 +
|pTet>>lox71-B0015(T)
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L47
 +
|lox71-B0015(T) BI (D57.2)
 +
|15
 +
|araC/pBad-J61002 plasmid SP dig.(D41)
 +
|2
 +
|araC/pBad>>lox71-B0015(T)
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L48
 +
|lox71-gfp(tripart) BI (D37.1)
 +
|10
 +
|<bbpart>pJ23100</bbpart> SP(D1)
 +
|2
 +
|pJ23100>>lox71-gfptripart
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L49
 +
|lox71-gfp(tripart) BI (D37.1)
 +
|10
 +
|<bbpart>pJ23107</bbpart> SP(D3)
 +
|2
 +
|pJ23107>>lox71-gfptripart(T)
|
|
|}
|}

Revision as of 12:17, 20 September 2007

Contents

DapA Wanner deletion: pKD13-DapA homologies PCR

We diluted 1/100 overnight culture at 30°C of MG1655 transformed by pKD46: 50µL into 5mL LB ampicilline.
At OD = 0.1, we induced pKD46 expression by addition of arabinose (10mM final).
At OD = 0.4, we prepared cells to be electrocompetent. (see Protocol)
Then we transformed the bacteria with PCR product from yesterday (see September 17).

Minipreps

Minipreps sur cultures ON de:

  • S24: (lox66-DapAsubtilis) MPT3.? (préciser David)
  • S26: (lRBS-DapAsubtilis) MPT5.? (préciser David)
  • pKD13: (plasmide FRT-KanR-FRT de Wanner), un stock glycerol est également réalisé

Digestion reactions

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D77 araC/pBad>>lox71-gfptripart BI D60 (.1 & .2) digestion product FspI AhdI in D60, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion
D78 araC/pBad>>lox71-gfptripart FI D71 (.1 & .2) digestion product FspI AhdI in D71, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion
D53 RBS-dapAsubtilis BBinsert pTOPO of RBS-dapAsubtilis (MPT5.2) EcoRI Pst1 RBS-dapAsubtilis extracted for cloning as a biobrick
D54 RBS-dapAsubtilis BI pTOPO of RBS-dapAsubtilis (MPT5.2) XbaI Pst1 RBS-dapAsubtilis extracted for use as a BI


Ligation reactions

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
C10 gfptripart-lox66 BV pSB1A2 (D69) 2
C11 B0015(T)-lox66 BV (D58.1) 3
C12 attB BV (D68) 2
C13 FRT-CmR-FRT BBa_J61025 BV (D64) 1.5
C14 lox66-mRFP FV (D61.1) 1.5
C15 B0015 BV pSB1AK3 (D46) 1
C16 pSB2K3 ES open vector (D73) 4
C17 pSB2K3 BBvector (D72) 2
C18 pSB1A2 EP open vector (D22) 2
L44 lox71-B0015(T) BI (D57.1) 15 pJ23100 SP(D1) 2 pJ23100>>lox71-B0015(T)
L45 lox71-B0015(T) BI (D57.1) 15 pJ23107 SP(D3) 2 pJ23107>>lox71-B0015(T)
L46 lox71-B0015(T) BI (D57.2) 15 BBa_J61002 pTet SP dig.(D50) 2 pTet>>lox71-B0015(T)
L47 lox71-B0015(T) BI (D57.2) 15 araC/pBad-J61002 plasmid SP dig.(D41) 2 araC/pBad>>lox71-B0015(T)
L48 lox71-gfp(tripart) BI (D37.1) 10 pJ23100 SP(D1) 2 pJ23100>>lox71-gfptripart
L49 lox71-gfp(tripart) BI (D37.1) 10 pJ23107 SP(D3) 2 pJ23107>>lox71-gfptripart(T)
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