Difference between revisions of "Template:IONIS Paris/Notebook/14-09-15"

PCR Nanoluc

Mix PCR preparation:

Tube	pSB1C3-Gblock
MQ Water	40 µL
RB Buffer	5 µL
Mg2+	1,5 µL
dNTP 10 µM	1 µL
Primer Fwd	0,5 µL
Primer Rev	0,5 µL
DNA	1µL
Taq Pol	0,5 µL

PCR cycle – IGEM TAQ NanoLuc

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	58	30 sec
Extension	72	1 min
Final extension	72	5 min	1
Hold	4	Infinite

PCR colony for pSB1C3-VVD YC + YN and pSB1C3-VVD YN + YC

Mix PCR preparation:

Tube	pSB1C3-VVD YC + YN (x7)	pSB1C3-VVD YN + YC (x6)
MQ Water	40 µL	40 µL
RB Buffer	5 µL	5 µL
Mg2+	1,5 µL	1,5 µL
dNTP 10 µM	1 µL	1 µL
Primer Fwd	YC Fwd, 0,5 µL	pSB1C3 Fwd 2, 1 µL
Primer Rev	pSB1C3 Rev 2, 1 µL	YN Rev 1, 0,5 µL
Taq Pol	0,5 µL	0,5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	35
Annealing	51	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Not expected bands for every PCR colony Could be due to the choice of primers

• Make another one with primers for pSB1C3 (more simple)

Digestion

Mix digestion preparation:

Tube	pSB1C3-pDawn	pSB1C3 iGEM	Nanoluc
H2O MQ	-	-	14 μL
Buffer 2.1	1,2 μL	1,1 μL	2 μL
DNA	10 μL	8 μL	2 μL
Enzyme 1	XbaI 1 μL	EcoRI 1 μL	EcoRI 1 μL
Enzyme 2	-	PstI 1 μL	PstI 1 μL

37°C, 1h heat kill: 80°C, 20min

Ligation

Mix ligation preparation:

Tube	pSB1C3-pDawn	pSB1C3-pDawn I-V (1:2)	pSB1C3-pDawn I-V (2:1)	pSB1C3-nanoluc (1:1)	pSB1C3-nanoluc (1:2)
H2O MQ	-	-	-	0,8 μL	4,3 μL
T4 buffer	1,4 μL	-	0,5 μL	0,5 μL	1 μL
pSB1C3	-	1,4 µL	5,5 µL	7 µL	3,5 µL
Insert	12,2 μL	20 μL	22 μL	0,7 μL	0,7 μL
T4 ligase	0,5 μL	1 μL	1 μL	0,5 μL	0,5 μL

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

• Transformation of competent cells with all ligation products using 1 µL or 3 µL

PCR colony for pSB1C3-VVD YC + YN and pSB1C3-VVD YN + YC

Mix PCR preparation:

Tube	pSB1C3-VVD YC + YN (x7)	pSB1C3-VVD YN + YC (x7)
MQ Water	40 µL	40 µL
RB Buffer	5 µL	5 µL
Mg2+	1,5 µL	1,5 µL
dNTP 10 µM	1 µL	1 µL
Primer pSB1C3 Fwd	1 µL	1 µL
Primer pSB1C3 Rev	1 µL	1 µL
Taq Pol	0,5 µL	0,5 µL

No differences with previous PCR colony
Bands weight do not correspond to the two possible cases “no insertion” or “insertion” of the part

• Liquid culture of 1 colony of each to analyse the DNA
  

Bacterial growth kinetic of bacteria transformed with pDawn-H/E

Liquid culture of 100 ml LB + 100 µl Kan for 5 interesting colonies from the experiment of the 09/09
37°C, 120 rpm, O/N, no light exposition

14 Sept. 15