Paris/September 17
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| + | |} | ||
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| + | ==Ligations== | ||
| + | {| | ||
| + | |- style="background: #ccccff;" | ||
| + | ! colspan="5" style="background: #ccccff;" | Ligations | ||
| + | |- style="background: #ccccff; text-align: center;" | ||
| + | |width=5%| Number | ||
| + | |width=20%| Insert | ||
| + | |width=5%| Insert Volume (µL) | ||
| + | |width=25%| Vector | ||
| + | |width=5%| Vector Volume (µL) | ||
| + | |width=35%| Comments | ||
| + | |width=5%| Number of colonies | ||
| + | |- style="background: #cccccc;" | ||
| + | | style="background: #ccffcc;" |C1 | ||
| + | | | ||
| + | | | ||
| + | |MP3.1 digested with EcoRI (D0) | ||
| + | |2 | ||
| + | |control of ligation efficiency | ||
| | | | ||
|} | |} | ||
Revision as of 17:05, 17 September 2007
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)
- 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 | |||
