Template:IONIS Paris/Notebook/30-07-15

pDawn amplification and digestion

PCR2 amplification of pDawn III results

Expected results

Results

Little amplification but not enough

PCR pDawn III

Mix PCR preparation:

	pDawn III	pDawn III
MQ water	40 µL	26,5 µL
RB Buffer	5 µL	/
Mg2+	1,5 µL	/
Q5 Buffer	/	10 µL
High GC content Buffer	/	10 µL
dNTPs 10µM	1 µL	1 µL
Primer Fwd 50µM	0,5 µL pDawn Fwd VI	0,5 µL pDawn Fwd VI
Primer Rev 50µM	0,5 µL pDawn Rev III-VI	0,5 µL pDawn Rev III-VI
DNA	1µL pDawn III PCR1	1µL pDawn III PCR1
Taq Pol	0.5 µL	/
Q5 Pol	/	0.5 µL

PCR cycle – IGEM TAQ

	T°C	Time	Cycle
Initial denaturation	98	5 min	1
Denaturation	98	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

Expected results

Results

Very good amplification of pDawn III
Gel extraction (kit from QIAgen)

Digestion of pDawn I, II and III

Mix digestion preparation:

Tube	pDawnI				pDawnII				pDawnIII
Water	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL	16 µL
Buffer 2.1	2 µL	2 µL	2 µL	/	2 µL	2 µL	2 µL	/	2 µL	2 µL	2 µL	/
Buffer 3.1	/	/	/	2 µL	/	/	/	2 µL	/	/	/	2 µL
DNA	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL	1,5 µL
Enzyme EcoRI	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5µL	/	/	/
Enzyme XbaI	/	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5 µL	/	/
Enzyme SpeI	/	/	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5 µL	/
Enzyme PstI	/	/	/	0,5 µL	/	/	/	0,5 µL	/	/	/	0,5 µL

37°C, 60 min

Expected results

Results

Simple digestions have shown that no other unexpected restriction site have appeared during PCR amplification of each fragment

30 July 15