Template:IONIS Paris/Notebook/28-07-15

Results of the last transformation

2 red colonies (which is weird)
Liquid culture for amplification

PCR fragments quantification

Expected results

Results

We get expected bands but, as for second PCRs of VVD and YN155 fragments, bands 2 time heavier than expected bands appeared

Test PCR pDawn (whole fragment)

Mix PCR preparation:

	pDawn (x3)	YC 155
MQ water	26,5 µL	26,5 µL
Q5 Buffer	10 µL	10 µL
High GC content Buffer	10 µL	10 µL
dNTPs 10µM	1 µL	1 µL
Primer Fwd 50µM	0,5 µL pDawn Fwd I	0,5 µL YC155 Fwd
Primer Rev 50µM	0,5 µL pDawn Rev III-VI	0,5 µL YC155 Rev
DNA	1µL pDawn	1µL YC155 PCR1
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	17
Annealing	59-60-61	30 sec
Extension	72	2 min
Final extension	72	10 min	1
Hold	4	Infinite

Expected results

Results

We get an amplification of pDawn independent of the temperature

Expected results

Results

After the last Gibson reaction we have get red colonies that should correspond to bacteria transformed with pSB1C3-RFP (which should be impossible after the gel migration and purification). So we analyzed the content of the gel extraction and we saw that the tube only had linearized backbone. About pDawn III PCR 2, the migration of 5 µl from the gel extraction of PCR products have shown that there is not a high quantity of DNA material, not enough for a Gibson assembly…

28 July 15