Paris/July 25

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==Digestions=
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[[Paris/July 24|yesterday]] -- [[Paris/July 26|tomorrow]] <br>
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==Digestions==
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* P8 (RBS-DapAColi) with XbaI & PstI for BW insert
+
-
                                      EcoRI & PstI for BioBricks
+
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* P9 (RBS-DapASubtilis) with XbaI & PstI for BW insert
+
-
                                            EcoRI & PstI for BioBricks
+
-
* P10 (Lox71-FtsA-FtsZ) with XbaI & PstI for BW insert
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 +
Digestion of purifications of PCR products<br>
{|
{|
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|width=35%| Description
|width=35%| Description
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |D22
+
| style="background: #ccffcc;" |D28
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|pSB1A2 open vector
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|RBS-DapAColi BB
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|<bbpart>BBa_J61047</bbpart> (MP9.1 & MP9.2)
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|P8 (RBS-DapAColi)
-
|EcoI
+
|EcoRI
|PstI
|PstI
-
|
+
|~950
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|Open pSB1A2 vector for BioBrick EcoRI/PstI insertio
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|Biobrick
|
|
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |D23
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| style="background: #ccffcc;" |D29
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|AraC/pBad promoter fw-insert
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|RBS-DapAColi
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|<bbpart>BBa_I0500</bbpart> (MP4.1 & MP4.2)
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|P8 (RBS-DapAColi)
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|EcoI
+
-
|SpeI
+
-
|
+
-
|AraC/pBad promoter ready for use as a forward insert
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-
|
+
-
|
+
-
|- style="background: #cccccc;" 
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-
| style="background: #ccffcc;" |D24
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-
|eCFP bw-insert
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-
|<bbpart>BBa_E0422</bbpart> (MP6.1 & MP6.2)
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|XbaI
|XbaI
|PstI
|PstI
-
|
+
|~950
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|eCFP (RBS-OFR_LVA-T) ready for use as a backward insert
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|Backward insert
|
|
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |D25
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| style="background: #ccffcc;" |D30
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|PoPs to GFP converter
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|RBS-DapASubtilis BB
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|<bbpart>BBa_E0421</bbpart> (MP7.1 & MP7.2)
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|P9 (RBS-DapASubtilis)
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|XbaI
+
|EcoRI
|PstI
|PstI
-
|
+
|~950
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|PoPs to GFP converter ready for use as a backward insert
+
|Biobrick
|
|
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |D26
+
| style="background: #ccffcc;" |D31
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|gfp-tripart
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|RBS-DapASubtilis
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|<bbpart>BBa_E0840</bbpart> (MP8.1 & MP8.2)
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|P9 (RBS-DapASubtilis)
|XbaI
|XbaI
|PstI
|PstI
-
|
+
|~950
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|gfp-tri part (strongRBS-ORF-T) ready for use as a backward insert
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|Backward insert
|
|
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
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| style="background: #ccffcc;" |D27
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| style="background: #ccffcc;" |D32
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|lox71-ftsZ bw-insert
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|Lox71-FtsA-FtsZ
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|lox71-ftsZ PCR product P7
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|P10 Lox71-FtsA-FtsZ
|XbaI
|XbaI
|PstI
|PstI
-
|
+
|~2600
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|lox71-ftsZ(unmutated) ready for use as a backward insert
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|Backward insert - EcoRI site still presents : we can't use EcoRI
|
|
|
|
 +
|}
 +
 +
 +
== Ligation & transformation reactions ==
 +
 +
 +
The Ligation reactions performed yesterday have all failed (the reactions were performed at 42° by mistake, then, another 1µL of ligase was added for a 5min rxn at 25°)
 +
 +
The following reactions were performed today
 +
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Ligations
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
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|width=25%| Insert
 +
|width=5%| Insert Volume (µL)
 +
|width=25%| Vector
 +
|width=5%| Vector Volume (µL)
 +
|width=35%| Comments
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L1'
 +
|D23.1 (AraC/pBad promoter FI)
 +
|8.5
 +
|D9.1 (J61002 ready for insertion of a FI)
 +
|1.5
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|construct with pBad promotor regulating expression of mRFP (in order to test activity of pBad)
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L2'
 +
|D18 (BB dig. lox66DapAE.coli)
 +
|7
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|D22.1 (pSB1A2 Eco, Pst)
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|3
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|Insertion of lox66DapAE.coli construct as a biobrick in pSB1A2 plasmid
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|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L3'
 +
|D19 (BB dig. lox66DapAsubtilis)
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|7
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|D22.1 (pSB1A2 Eco, Pst)
 +
|3
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|Insertion of lox66DapAsubtilis construct as a biobrick in pSB1A2 plasmid
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L4'
 +
|D20 (Lox66-DapAE.coli BI)
 +
|8
 +
|D1 (pJ23100 BV)
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|2
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|lo66-DapAE.coli under regulation of strong promotor pJ23100
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L5'
 +
|D21 (lox66-DapAsubtilis BI)
 +
|6.5
 +
|D1 (pJ23100 BV)
 +
|2 (+1.5µL H2O)
 +
|lox66-DapAsubtilis under regulation of strong promotor pJ23100
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L6'
 +
|D14.1 (Cre ORF)
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|8
 +
|D15.1 (B0030 BV)
 +
|2
 +
|construction of the biobrick strong RBS(B0030)-Cre ORF
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L7'
 +
|D27 (Lox71-ftsZ BI)
 +
|6
 +
|D1 (pJ23100 BV)
 +
|2 (+2µL H2O)
 +
|lox71-ftsZ under regulation of strong promotor pJ23100
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L8'
 +
|D27 (Lox71-ftsZ BI)
 +
|6
 +
|D3 (pJ23107 BV)
 +
|2 (+2 H2O)
 +
|lox71-ftsZ under regulation of promotor pJ23107 (intermediate strength)
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L9'
 +
|lox66 [O14+O15] (0.2µM)
 +
|2
 +
|D22.1 (pSB1A2 Eco, Pst)
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|2 (+6 H2O)
 +
|lox66 cloned as a biobrick in pSB1A2
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|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L10'
 +
|lox71 [O16+O17] (0.2µM)
 +
|2
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|D22.1 (pSB1A2 Eco, Pst)
 +
|2 (+6 H2O)
 +
|lox71 cloned as a biobrick
|}
|}

Latest revision as of 17:50, 7 October 2007

yesterday -- tomorrow

Digestions

Digestion of purifications of PCR products

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 size Description
D28 RBS-DapAColi BB P8 (RBS-DapAColi) EcoRI PstI ~950 Biobrick
D29 RBS-DapAColi P8 (RBS-DapAColi) XbaI PstI ~950 Backward insert
D30 RBS-DapASubtilis BB P9 (RBS-DapASubtilis) EcoRI PstI ~950 Biobrick
D31 RBS-DapASubtilis P9 (RBS-DapASubtilis) XbaI PstI ~950 Backward insert
D32 Lox71-FtsA-FtsZ P10 Lox71-FtsA-FtsZ XbaI PstI ~2600 Backward insert - EcoRI site still presents : we can't use EcoRI


Ligation & transformation reactions

The Ligation reactions performed yesterday have all failed (the reactions were performed at 42° by mistake, then, another 1µL of ligase was added for a 5min rxn at 25°)

The following reactions were performed today

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments
L1' D23.1 (AraC/pBad promoter FI) 8.5 D9.1 (J61002 ready for insertion of a FI) 1.5 construct with pBad promotor regulating expression of mRFP (in order to test activity of pBad)
L2' D18 (BB dig. lox66DapAE.coli) 7 D22.1 (pSB1A2 Eco, Pst) 3 Insertion of lox66DapAE.coli construct as a biobrick in pSB1A2 plasmid
L3' D19 (BB dig. lox66DapAsubtilis) 7 D22.1 (pSB1A2 Eco, Pst) 3 Insertion of lox66DapAsubtilis construct as a biobrick in pSB1A2 plasmid
L4' D20 (Lox66-DapAE.coli BI) 8 D1 (pJ23100 BV) 2 lo66-DapAE.coli under regulation of strong promotor pJ23100
L5' D21 (lox66-DapAsubtilis BI) 6.5 D1 (pJ23100 BV) 2 (+1.5µL H2O) lox66-DapAsubtilis under regulation of strong promotor pJ23100
L6' D14.1 (Cre ORF) 8 D15.1 (B0030 BV) 2 construction of the biobrick strong RBS(B0030)-Cre ORF
L7' D27 (Lox71-ftsZ BI) 6 D1 (pJ23100 BV) 2 (+2µL H2O) lox71-ftsZ under regulation of strong promotor pJ23100
L8' D27 (Lox71-ftsZ BI) 6 D3 (pJ23107 BV) 2 (+2 H2O) lox71-ftsZ under regulation of promotor pJ23107 (intermediate strength)
L9' lox66 [O14+O15] (0.2µM) 2 D22.1 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox66 cloned as a biobrick in pSB1A2
L10' lox71 [O16+O17] (0.2µM) 2 D22.1 (pSB1A2 Eco, Pst) 2 (+6 H2O) lox71 cloned as a biobrick
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