Paris/September 17

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DapA Wanner deletion: pKD13 - DapA homologies PCR

  • Material:

- pKD13: 5 µL (bacteria)
- Tampon GC: 10µL
- dNTP: 1µL (final 200µmol each)
- Taq: 0.5µL
- Water: 32.5µL
- Primer1: 33: 2.5µL (final 0.5µM)
- Primer2: 34: 2.5µL (final 0.5µM)

File:WannerDapA09172007.jpg

  • PCR Program:

35 cycles:
- 98°C: 5min
- 98°C: 45s (X35)
- 69°C: 30s (X35)
- 72°C: 1min (X35)
- 72°C: 10 min
- 4°C: inf

  • Quantity: ng/µL
  • Expected size: 1403 kb

Digestions

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D56 araC/pBad>>RBS-Cre BBinsert L26.1 araC/pbad>>RBS-Cre in J61002 EcoRI Pst1 araC/pBad>>RBS-Cre BBinsert (for transfer to the low-copy plasmid vector)
D66 KanR BBa_J61025 FV (MP12.1, MP12.2) BBa_P1003 in pSB1A2 EcoR XbaI KanR BBa_J61025 (for forward introduction of a casette for Wanner K insertion)
D71 araC/pBad>>lox71-gfptripart FI L35.1 araC/pBad>>lox71-gfptripart BBa_J61002 EcoRI SpeI araC/pBad>>lox71-gfptripart for use as a FI
D72 pSB2K3 BBvector MP16 pSB2K3::attP EcoRI PstI Low copy number plasmid, BB vector
D73 pSB2K3 BBvector MP16 pSB2K3::attP EcoRI SpeI Comparison of digestion efficiency between Pst and Spe in NEB2
D75 AraC-pBAD-RBS-CRE FI L26.1 AraC-pBAD-RBS-CRE EcoRI SpeI FI, Comparison of digestion efficiency between Pst and Spe in NEB2 (see D56)
D76 DGAT BB P14 DGAT EcoRI PstI DGAT

Ligations

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
C1 MP3.1 digested with EcoRI (D0) 2 control of ligation efficiency