Paris/September 17

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Revision as of 15:22, 18 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)

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

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

@@ Line 88: / Line 88: @@
 |- style="background: #cccccc;"
 | style="background: #FF99FF;" |D73
-|<bbpart>pSB2K3</bbpart> BBvector
+|<bbpart>pSB2K3</bbpart> ES open vector
 |MP16 <bbpart>pSB2K3</bbpart>::attP
 |EcoRI