Paris/July 24


yesterday -- tomorrow



BBa_E0422 in pSB1A2 - Clone 1&2 - MP6.1' & MP6.2'
BBa_E0241 in pSB1A2 - Clone 1 - MP7.1'
BBa_J61047 in pSB1A2 - Clone 1&2 - MP9.1' & MP9.2'

Ligation & transformation reactions

Number Insert Insert Volume Vector Vector Volume Comments
L1 D23 (AraC/pBad promoter FI) D9 (J61002 ready for insertion of a FI)
L2 D18 (BB dig. lox66DapAE.coli) D22 (pSB1A2 Eco, Pst)
L3 D19 (BB dig. lox66DapAsubtilis) D22 (pSB1A2 Eco, Pst)
L4 D20 (Lox66-DapAE.coli BI) D1 (pJ23100 BV)
L5 D21 (lox66-DapAsubtilis BI) D1 (pJ23100 BV)
L6 D20 (Lox66-DapAE.coli BI) D3 (pJ23107 BV)
L7 D21 (lox66-DapAsubtilis BI) D3 (pJ23107 BV)
L8 D14 (Cre ORF) D15 (B0030 BV)
L9 D27 (Lox71-ftsZ BI) D1 (pJ23100 BV)
L10 D27 (Lox71-ftsZ BI) D3 (pJ23107 BV)
  • lox66 (annealing O14 & O15) & lox71 (annealing O16 & O17) in D22 (pSB1A2 Eco+Pst)

Annealing of Lox66 and Lox71

Lox66 and Lox71 oligos were designed to form a double-stranded DNA. The extremities bear cohesive overhangs corresponding to digestion by EcoRI and SpeI. See oligos 14 + 15 and 16 + 17 here.

Annealing mix:

  • 8 μL of each of the concentrated primers
  • 4 μL of salt solution (10 mM NaCl)
  • 20 μL of water

Mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.
More information here

Purification of PCR Products

  • RBS-DapAColi = P8
  • RBS-DapASubtilis = P9
  • Lox71-FtsA-FtsZ = P10
  • DGAT1 = P3
  • DGAT2 = P4