Paris/September 14

From 2007.igem.org

yesterday -- tomorrow

DapA Wanner deletion: pKD3/pKD4-DapA homologies PCR

  • Material:

- pKD3/pKD4: 1 µL (1.5ng/3ng)
- 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:WannerDapA09142007.jpg

FAILED

  • PCR Program:

35 cycles:
- 98°C: 5min
- 98°C: 30s (X35)
- 69°C: 30s (X35)
- 72°C: 1min30 (X35)
- 72°C: 10 min
- 4°C: inf

  • Quantity: ng/µL (pKD3) et ng/µL (pKD4)
  • Expected size: 1.1 kb (pKD3) / 1.6 kb (pKD4)

PCRs have failed for 4 days because the primers are designed for pKD13.

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)
D57 lox71-B0015(T) BI (L31.1, L31.3) lox71-B0015(T) pSB1A2 XbaI Pst1 lox71-B0015(T) extracted for use as a BI pSB1A2
D58 B0015(T)-lox66 BV (L32.1, L32.2) B0015(T)-lox66 BV SpeI PstI B0015(T)-lox66 for use as a BV pSB1A2
D59 pTet>>lox71-gfptripart BI (L34.1, L34.2) pTet>>lox71-gfptripart BBa_J61002 XbaI PstI pTet>>lox71-gfptripart for use as a BI
D60 araC/pBad>>lox71-gfptripart BI (L35.1, L35.2) araC/pBad>>lox71-gfptripart BBa_J61002 XbaI PstI araC/pBad>>lox71-gfptripart for use as a BI
D61 lox66-mRFP FV (L36.1, L36.2) lox66-mRFP EcoRI XbaI lox66-mRFP part (strongRBS-ORF-T) ready for use as a forward vector
D62 lox66-RBSDapAcoli BI (L37.1, L37.2) lox66-RBSDapAcoli XbaI PstI lox66-RBSDapAcoli ready for use as a Backward insert
D63 RBSDapAcoli BI (L39.1, L39.2) RBSDapAcoli XbaI PstI RBSDapAcoli BI (for insertion behind T-lox66)
D64 FRT-CmR-FRT BBa_J61025 BV (MP14.1) BBa_J61025 SpeI PstI FRT-CmR-FRT BBa_J61025 (for backward introduction of a casette for Wanner K insertion)
D65 FRT-CmR-FRT BBa_J61025 FV (MP14.1) BBa_J61025 EcoR XbaI FRT-CmR-FRT BBa_J61025 (for forward introduction of a casette for Wanner K insertion)
D66 KanR BBa_J61025 FV (MP12.1) BBa_P1003 in pSB1A2 EcoR XbaI KanR BBa_J61025 (for forward introduction of a casette for Wanner K insertion)
D67 RBS-DapAsubtilis BI (L43.1, L43.2) RBS-DapAsubtilis pSB1A2 XbaI PstI RBS-DapAsubtilis for use as a BI
D68 attB BV (MP15.1) SpeI PstI vector containing attB for use as a BV
D69 gfptripart-lox66 BV (L29.1) gfptripart-lox66 pSB1A2 SpeI PstI
D70 pTet>>lox71-gfptripart FI (L34.1, L34.2) pTet>>lox71-gfptripart BBa_J61002 EcoRI SpeI pTet>>lox71-gfptripart for use as a FI
D71 araC/pBad>>lox71-gfptripart FI (L35.1, L35.2) araC/pBad>>lox71-gfptripart BBa_J61002 EcoRI SpeI araC/pBad>>lox71-gfptripart for use as a FI