Paris/July 25

From 2007.igem.org

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