Paris/July 25

From 2007.igem.org

< Paris(Difference between revisions)
(Ligation & transformation reactions)
 
(4 intermediate revisions not shown)
Line 1: Line 1:
 +
[[Paris/July 24|yesterday]] -- [[Paris/July 26|tomorrow]] <br>
==Digestions==
==Digestions==
Line 85: Line 86:
|width=35%| Comments
|width=35%| Comments
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L1
+
| style="background: #ccffcc;" |L1'
|D23.1 (AraC/pBad promoter FI)
|D23.1 (AraC/pBad promoter FI)
|8.5
|8.5
|D9.1 (J61002 ready for insertion of a FI)
|D9.1 (J61002 ready for insertion of a FI)
|1.5
|1.5
-
|
+
|construct with pBad promotor regulating expression of mRFP (in order to test activity of pBad)
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L2
+
| style="background: #ccffcc;" |L2'
|D18 (BB dig. lox66DapAE.coli)
|D18 (BB dig. lox66DapAE.coli)
|7
|7
|D22.1 (pSB1A2 Eco, Pst)
|D22.1 (pSB1A2 Eco, Pst)
|3
|3
-
|
+
|Insertion of lox66DapAE.coli construct as a biobrick in pSB1A2 plasmid
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L3
+
| style="background: #ccffcc;" |L3'
|D19 (BB dig. lox66DapAsubtilis)
|D19 (BB dig. lox66DapAsubtilis)
|7
|7
|D22.1 (pSB1A2 Eco, Pst)
|D22.1 (pSB1A2 Eco, Pst)
|3
|3
-
|
+
|Insertion of lox66DapAsubtilis construct as a biobrick in pSB1A2 plasmid
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L4
+
| style="background: #ccffcc;" |L4'
|D20 (Lox66-DapAE.coli BI)
|D20 (Lox66-DapAE.coli BI)
|8
|8
|D1 (pJ23100 BV)
|D1 (pJ23100 BV)
|2
|2
-
|
+
|lo66-DapAE.coli under regulation of strong promotor pJ23100
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L5
+
| style="background: #ccffcc;" |L5'
|D21 (lox66-DapAsubtilis BI)
|D21 (lox66-DapAsubtilis BI)
|6.5
|6.5
|D1 (pJ23100 BV)
|D1 (pJ23100 BV)
|2 (+1.5µL H2O)
|2 (+1.5µL H2O)
-
|
+
|lox66-DapAsubtilis under regulation of strong promotor pJ23100
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L6
+
| style="background: #ccffcc;" |L6'
|D14.1 (Cre ORF)
|D14.1 (Cre ORF)
|8
|8
|D15.1 (B0030 BV)
|D15.1 (B0030 BV)
|2
|2
-
|
+
|construction of the biobrick strong RBS(B0030)-Cre ORF
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L7
+
| style="background: #ccffcc;" |L7'
|D27 (Lox71-ftsZ BI)
|D27 (Lox71-ftsZ BI)
|6
|6
|D1 (pJ23100 BV)
|D1 (pJ23100 BV)
|2 (+2µL H2O)
|2 (+2µL H2O)
-
|
+
|lox71-ftsZ under regulation of strong promotor pJ23100
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L8
+
| style="background: #ccffcc;" |L8'
|D27 (Lox71-ftsZ BI)
|D27 (Lox71-ftsZ BI)
|6
|6
|D3 (pJ23107 BV)
|D3 (pJ23107 BV)
|2 (+2 H2O)
|2 (+2 H2O)
-
|
+
|lox71-ftsZ under regulation of promotor pJ23107 (intermediate strength)
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L9
+
| style="background: #ccffcc;" |L9'
|lox66 [O14+O15] (0.2µM)
|lox66 [O14+O15] (0.2µM)
|2
|2
|D22.1 (pSB1A2 Eco, Pst)
|D22.1 (pSB1A2 Eco, Pst)
|2 (+6 H2O)
|2 (+6 H2O)
-
|
+
|lox66 cloned as a biobrick in pSB1A2
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |L10
+
| style="background: #ccffcc;" |L10'
|lox71 [O16+O17] (0.2µM)
|lox71 [O16+O17] (0.2µM)
|2
|2
|D22.1 (pSB1A2 Eco, Pst)
|D22.1 (pSB1A2 Eco, Pst)
|2 (+6 H2O)
|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