Template:IONIS Paris/Notebook/01-09-15

Digestion pDawn and pDawn Gblocks (I – V)

Mix digestion preparation:

Tube	pDawn	pDawn I	pDawn II	pDawn III	pDawn IV	pDawn V
Buffer (2 µL)	Buffer 2.1	Buffer 3.1	Cutsmart	Cutsmart	Buffer 2.1	Buffer 2.1
DNA	12 µL	12 µL	12 µL	12 µL	12 µL	12 µL
Enzyme 1 (0,5 µL)	EcoRI	SalI HF	SalI HF	XmaI	SphI	SphI
Enzyme 2 (0,5 µL)	PstI	-	XmaI	SphI	-	-

37°C, 1h heat kill: 80°C, 20min

Expected results

Results

pDawn quantification = 22,8 ng.µL^-1

Ligation of Gblocks (GFP, H/E, CCDB, HOKD) into pDawn

Mix ligation preparation:

Tube	GFP	H/E	ccdB	HokD
Water	5,1 µL	4,6 µL	4,8 µL	5,3 µL
T4 ligase Buffer	1 µL	1 µL	1 µL	1 µL
pDawn	0,9 µL	1,4 µL	1,2 µL	0,7 µL
Plasmid	2,5 µL	2,5 µL	2,5 µL	2,5 µL
T4 ligase	0,5 µL	0,5 µL	0,5 µL	0,5 µL

Room temperature, 30min heat kill: 80°C, 20min

Transformation of E.coli DH5 alpha competent cells

Dephosphorylation of pDawn IV

Mix plasmid dephosphorylation preparation:

Tube	GFP
Cutsmart Buffer	2 µL
Plasmid	15 µL
rSAP enzyme	1 µL

Ligation of pDawn Gblocks

Mix ligation preparation:

Tube	pDawn I-II-III	pDawn IV-V
Water	4,3 µL	3,7 µL
T4 ligase Buffer	1 µL	1 µL
pDawn I	0,9 µL	-
pDawn II	2,3 µL	-
pDawn III	2,3 µL	-
pDawn IV	-	2,5 µL
pDawn IV	-	2,5 µL
T4 ligase	0,5 µL	0,5 µL

Room temperature, 30min heat kill: 65°C, 20 min

1 September 15