Paris/September 14
From 2007.igem.org
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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)
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 | ||||
