From 2007.igem.org

yesterday -- tomorrow

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 ES open vector	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