Difference between revisions of "Template:IONIS Paris/Notebook/11-09-15"

Digestion

Mix digestion preparation:

Tube	VVD YC	VVD YN
H2O MQ	1 μL	1 μL
Buffer 2.1	2 μL	2 μL
DNA	15 μL	15 μL
Enzyme 1	EcoRI 1 μL	XbaI 1 μL
Enzyme 2	SpeI 1 μL	PstI 1 μL

37°C, 1h heat kill: 65°C, 20min

Ligation

Mix ligation preparation:

Tube	pSB1C3-VVD YC	pSB1C3-VVD YN	pSB1C3
H2O MQ	2,2 μL	3,4 μL	1,8 μL
T4 ligase buffer	1 μL	1 μL	2 μL
Insert	VVD YN, 4,1 µL	VVD YC, 2,9 µL	pDawn (28/07), 7,2 µL
Backbone	2,2 μL	2,2 μL	8 μL
T4 ligase	0,5 μL	0,5 μL	1 μL

37°C, 30min heat kill: 65°C, 20min

• Transformation of competent cells with pSB1C3-VVD YC + VVD YN and pSB1C3-VVD YN + VVD YC (1 µL or 4 µL have been used); 30°C all the weekend long

Ligation pDawn Gblock

Mix ligation preparation:

Tube	pDawn I-II-III	pDawn IV-V
T4 ligase buffer	2 μL	2,3 μL
pDawn I	2,3 μL	-
pDawn II	12 μL	-
pDawn III	2,7 μL	-
pDawn IV	-	13,2 μL
pDawn V	-	6,2 μL
T4 ligase	1 μL	1 μL

Room temperature, 45min heat kill: 65°C, 20min

Purification of pSB1C3-pDawn

20 µL of ligation product + 80 µL of H₂O MQ
Add 1/10 (10 µL) of the volume of NaOAc 3M pH 5.2
Add 2,5 volumes (275 µL) of Ethanol 100%
Mix and stored at -20°C for 1 hour at least
Centrifuge 30 sec, 14000 rpm at 4°C
Discard the supernatant
Resuspend the pellet with 500 µL of ethanol 70%, invert up the tube several time
Centrifuge 5 min, 14000 rpm at 4°C
Discard the supernatant and let dry the pellet
Resuspend the pellet with the right amount of water following the final concentration expected (20 µL here)

Ligation pDawn Gblock

Mix ligation preparation:

Tube	pDawn I-II-III-IV-V
pDawn I-II-III	20 μL
pDawn IV-V	22,3 μL
T4 ligase	1 μL

Room temperature, 45min heat kill: 65°C, 20min

11 Sept. 15