Paris/September 13

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(→DapA Wanner deletion: pKD3/pKD4-DapA homologies PCR)
 
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[[Paris/September 12|yesterday]] -- [[Paris/September 14|tomorrow]]
[[Paris/September 12|yesterday]] -- [[Paris/September 14|tomorrow]]
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== Digestions ==
 
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{|
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== DapA Wanner deletion: pKD3/pKD4-DapA homologies PCR ==
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|- style="background: #ccccff;"
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! colspan="5" style="background: #ccccff;" | Digestion Products
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|- style="background: #ccccff; text-align: center;"
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|width=5%| Number
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|width=24%| Product Name
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|width=24%| 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=34%| Description
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|width=1%|
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|width=1%|
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|width=1%|
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|- style="background: #cccccc;" 
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-
| style="background: #FF99FF;" |D56
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|araC/pBad>>RBS-Cre BBinsert
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|L26.1 araC/pbad>>RBS-Cre in <bbpart>J61002</bbpart>
+
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|EcoRI
+
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|Pst1
+
-
|
+
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|araC/pBad>>RBS-Cre BBinsert (for transfer to the low-copy plasmid vector)
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-
|
+
-
|
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-
|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D57
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|lox71-BOO15(T) BI
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|L31.1, L31.3, L31.4 lox71-BOO15(T) <bbpart>pSB1A2</bbpart>
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|XbaI
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|Pst1
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-
|
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|lox71-BOO15(T) extracted for use as a BI <bbpart>pSB1A2</bbpart>
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|
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-
|
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-
|
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|- style="background: #cccccc;" 
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| style="background: #FF99FF;" |D58
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|B0015(T)-lox66 BV
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|L32.1, L32.2, L32.3 B0015(T)-lox66 BV
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-
|SpeI
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|PstI
+
-
|
+
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|B0015(T)-lox66 for use as a BV <bbpart>pSB1A2</bbpart>
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-
|
+
-
|
+
-
|
+
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|- style="background: #cccccc;" 
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-
| style="background: #ccffcc;" |D59
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|pTet>>lox71-gfptripart BI
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|L34.1, L34.2, L34.3 pTet>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
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|SpeI
+
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|PstI
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-
|
+
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|pTet>>lox71-gfptripart for use as a BI
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-
|
+
-
|
+
-
|
+
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|- style="background: #cccccc;" 
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-
| style="background: #ccffcc;" |D60
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|araC/pBad>>lox71-gfptripart BI
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|L35.1, L35.2 & L35.3 araC/pBad>>lox71-gfptripart <bbpart>BBa_J61002</bbpart>
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|SpeI
+
-
|PstI
+
-
|
+
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|araC/pBad>>lox71-gfptripart for use as a BI
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-
|
+
-
|
+
-
|
+
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|- style="background: #cccccc;" 
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-
| style="background: #FF99FF;"  |D61
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|lox66-mRFP FV
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|L36.1, L36.2 & L36.3 lox66-mRFP
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|EcoRI
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|XbaI
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|
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|lox66-gfptri part (strongRBS-ORF-T) ready for use as a forward vector
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|
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D62
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|lox66-RBSDapAcoli BI
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|L37.1, L37.2 & L37.3 lox66-RBSDapAcoli
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|XbaI
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|PstI
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|
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|lox66-RBSDapAcoli ready for use as a Backward insert
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|
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D63
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|RBSDapAcoli BI
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|L39.1, L39.2 & L39.3 RBSDapAcoli
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|XbaI
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|PstI
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-
|
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|RBSDapAcoli BI (for insertion behind T-lox66)
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|
+
-
|
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|
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|- style="background: #cccccc;" 
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| style="background: #FF99FF;" |D64
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|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> BV
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|MP14.1 <bbpart>BBa_J61025</bbpart>
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|SpeI
+
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|PstI
+
-
|
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|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> (for backward introduction of a casette for Wanner K insertion)
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|
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #FF99FF;" |D65
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|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> FV
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|MP14.1 <bbpart>BBa_J61025</bbpart>
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|EcoR
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|XbaI
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|
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|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> (for forward introduction of a casette for Wanner K insertion)
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|
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #FF99FF;" |D65
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|KanR <bbpart>BBa_J61025</bbpart> FV
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|MP12.1 <bbpart>BBa_P1003</bbpart> in <bbpart>pSB1A2</bbpart>
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|EcoR
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|XbaI
+
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|
+
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|KanR <bbpart>BBa_J61025</bbpart> (for forward introduction of a casette for Wanner K insertion)
+
-
|
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |D66
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|RBS-DapAsubtilis BI
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|L43.1, L43.2 & L43.3 RBS-DapAsubtilis <bbpart>pSB1A2</bbpart>
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|XbaI
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|PstI
+
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|
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|RBS-DapAsubtilis for use as a BI
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|
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|
+
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|
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|- style="background: #cccccc;" 
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| style="background: #FF99FF;" |D67
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|attB BV
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|MP15.1
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|SpeI
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|PstI
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|
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|vector containing attB for use as a BV
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-
|
+
-
|
+
-
|
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|- style="background: #cccccc;" 
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| style="background: #FF99FF;" |D68
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|gfptripart-lox66 BV
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|L29.1 gfptripart-lox66 <bbpart>pSB1A2</bbpart>
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|SpeI
+
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|PstI
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|
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|
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|
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|
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|
+
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|}
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digestion mix:
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We diluted 1/100 overnight culture at 30°C of MG1655 transformed by pKD46: 50µL into 5mL LB ampicilline.<br>
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* 25µl plasmid
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At OD = 0.1, we induced pKD46 expression by addition of arabinose (10mM final). <br>
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* 1µl BSA
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At OD = 0.4, we prepared cells to be electrocompetent. (see [[Paris/PROTOCOLS#Recombineering.2FLambda_red-mediated_gene_replacement|Protocol]])<br>
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* 10 µl NEB2
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Then we transformed the bacteria with PCR product from yesterday (see [[Paris/September_12|September 12]]).
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* 60µl H2O
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* 2µl Enzyme1
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== Sequencing ==
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* 2µl Enzyme2
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 +
The following plasmids were sent for sequencing rxn:
 +
 
 +
(name of the plasmid+oligo)
 +
 
 +
DB_L31.1+O18
 +
DB_L31.1+O19
 +
DB_L31.3+O18
 +
DB_L31.3+O19
 +
DB_L32.1+O18
 +
DB_L32.1+O19
 +
DB_L32.2+O18
 +
DB_L32.2+O19
 +
DB_L34.1+O18
 +
DB_L34.1+O19
 +
DB_L34.2+O18
 +
DB_L34.2+O19
 +
DB_L35.1+O18
 +
DB_L35.1+O19
 +
DB_L35.2+O18
 +
DB_L35.2+O19
 +
DB_L36.1+O18
 +
DB_L36.1+O19
 +
DB_L36.2+O18
 +
DB_L36.2+O19
 +
DB_L37.1+O18
 +
DB_L37.1+O19
 +
DB_L37.1+O26
 +
DB_L37.2+O18
 +
DB_L37.2+O19
 +
DB_L37.2+O26
 +
DB_L39.1+O18
 +
DB_L39.1+O19
 +
DB_L39.1+O26
 +
DB_L39.2+O18
 +
DB_L39.2+O19
 +
DB_L39.2+O26
 +
DB_L40.1+O18
 +
DB_L40.1+O19
 +
DB_L40.1+O25
 +
DB_L40.2+O18
 +
DB_L40.2+O19
 +
DB_L40.2+O25
 +
DB_L41.1+O18
 +
DB_L41.1+O19
 +
DB_L41.1+O24
 +
DB_L42.1+O18
 +
DB_L42.1+O19
 +
DB_L42.1+O24
 +
DB_L42.1+O22
 +
DB_L42.1+O23
 +
DB_L42.2+O18
 +
DB_L42.2+O19
 +
DB_L42.2+O24
 +
DB_L42.2+O22
 +
DB_L42.2+O23
 +
DB_L42.3+O18
 +
DB_L42.3+O19
 +
DB_L42.3+O24
 +
DB_L42.3+O22
 +
DB_L42.3+O23
 +
DB_L43.1+O18
 +
DB_L43.1+O19
 +
DB_L43.1+O25
 +
DB_L43.2+O18
 +
DB_L43.2+O19
 +
DB_L43.2+O25
 +
DB_L43.3+O18
 +
DB_L43.3+O19
 +
DB_L43.3+O25
 +
DB_MPT5.1+O41
 +
DB_MPT5.2+O41

Latest revision as of 14:50, 14 September 2007

yesterday -- tomorrow


DapA Wanner deletion: pKD3/pKD4-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 12).

Sequencing

The following plasmids were sent for sequencing rxn:

(name of the plasmid+oligo)

DB_L31.1+O18 DB_L31.1+O19 DB_L31.3+O18 DB_L31.3+O19 DB_L32.1+O18 DB_L32.1+O19 DB_L32.2+O18 DB_L32.2+O19 DB_L34.1+O18 DB_L34.1+O19 DB_L34.2+O18 DB_L34.2+O19 DB_L35.1+O18 DB_L35.1+O19 DB_L35.2+O18 DB_L35.2+O19 DB_L36.1+O18 DB_L36.1+O19 DB_L36.2+O18 DB_L36.2+O19 DB_L37.1+O18 DB_L37.1+O19 DB_L37.1+O26 DB_L37.2+O18 DB_L37.2+O19 DB_L37.2+O26 DB_L39.1+O18 DB_L39.1+O19 DB_L39.1+O26 DB_L39.2+O18 DB_L39.2+O19 DB_L39.2+O26 DB_L40.1+O18 DB_L40.1+O19 DB_L40.1+O25 DB_L40.2+O18 DB_L40.2+O19 DB_L40.2+O25 DB_L41.1+O18 DB_L41.1+O19 DB_L41.1+O24 DB_L42.1+O18 DB_L42.1+O19 DB_L42.1+O24 DB_L42.1+O22 DB_L42.1+O23 DB_L42.2+O18 DB_L42.2+O19 DB_L42.2+O24 DB_L42.2+O22 DB_L42.2+O23 DB_L42.3+O18 DB_L42.3+O19 DB_L42.3+O24 DB_L42.3+O22 DB_L42.3+O23 DB_L43.1+O18 DB_L43.1+O19 DB_L43.1+O25 DB_L43.2+O18 DB_L43.2+O19 DB_L43.2+O25 DB_L43.3+O18 DB_L43.3+O19 DB_L43.3+O25 DB_MPT5.1+O41 DB_MPT5.2+O41

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