Team:IONIS Paris/Notebook

Notebook

MARCH 2015

APRIL 2015

MAY 2015

JUNE 2015

JULY 2015

AUGUST 2015

SEPT. 2015

PCR pDawn BioBricks

PCR pDawn III

Aim: first step for pDawn amplification and mutagenesis

Mix PCR preparation:

	pDawn III
MQ water	39,5 µL
Mg2+ 50 mM	1,5 µL
Buffer RB	5 µL
dNTPs 10µL	1 µL
Primer Fwd 50µM	1µL pDawn Fwd III
Primer Rev 50µM	1µL pDawn Rev III
DNA	1µL pDawn
Taq Pol	0.5 µL

PCR cycle – IGEM TAQ 2

	T°C	Time	Cycle
Initial denaturation	95	30 sec	1
Denaturation	95	30 sec	17
Annealing	60	30 sec
Extension	72	2 min
Final extension	72	5 min	1
Hold	4	Infinite

Gibson Assembly

Aim: first step of our VVD Biobrick

Quantification of DNA fragments by Image J:

Fragments	Concentration (ng.µL-1)	Lenght	Molecular weight (g.mol-1)	Molarity (mol.L-1)	1/ratio	Dilution factor	Molarity after dilution	DNA Volume	Volume Water	Volume totale
pSB1C3	91.11	2 200	1 430 000	0.000000063712	7.75	1.35	0.000000047103	3.50	1.50	5
VVD1	15.71	250	162 500	0.000000096698	5.10	2.05	0.000000047103	1.00	1.00	2
VVD2	7.65	250	162 500	0.000000047103	10.48	1.00	0.000000047103	1.00	0	1
YC155	128.31	400	260 000	0.000000493488	1.00	10.48	0.000000047103	1.00	9.00	10

DNA mix:

• YC155: 1µL + 9µL of MQ water
• VVD1: 1µL + 1µL of MQ water
• VVD2: no dilution
• pSB1C3: 3.5µL + 1.5µL of MQ water

Mix = 2µL of each
5µl used for the Gibson assembly added to 15µl of master mix

Electrophoresis

Gel : 1,2 g agarose + 120 mL of TAE 1x + 150 µL of BET
Migration of pDawn III

Expected results

Results

Soft band at 1500 bp, good but not enough

1 July 15

Preparation of media

Aim: prepare media for bacteria culture

For 250mL

LB Broth	LB Agar
2.5g tryptone	2.5g tryptone
2.5g NaCl	2.5g NaCl
1.25g yeast extract	1.25g yeast extract
	5.0g agar

Qsp 250 ml distilled water
2 bottles of LB broth and 2 bottles of LB agar (+magnetic stirrer)
Auctoclave 121°C

28 March 15