Notebook

Day 1

Description of everything we do

Voici le titre du tableau

Titre A1	Titre A2	Titre A3	Titre A4
Titre B1	Valeur B2	Valeur B3	Valeur B4

02/06/2015

- Stock solution MgSO4 : 0,5M

Pour 500mL :

61,62g de MgSO4 (MM : 246,48)

qsp 500mL H2O stérile

- SOB Medium : 1 L

Final [c]	Composent	Volume & Mass
2%	Bactotryptone	20 g
10 mM	NaCl	2 mL (5 M stock)
2%	Yeast Extract	5 g
2,5 mM	KCl	0,18 g
10 mM	MgCl2	5 mL (2 M stock)
10 mM	MgSO4	20 mL (0,5 M)
-	H2O	qsp 1L
pH media to 7 with NaOH, then autoclave

- 2XTY Medium 500 mL

Composent	Volume & Mass
Bactotryptone	8 g
NaCl	2,5 g
Yeast Extract	5 g
H2O	qsp 500 mL

03/06/2015

- Transformation Broth (TB): 1 L

Final [c]	Composent	Volume & Mass
10 mM	Pipes	3,02 g
15 mM	CaCl2	2,21 g
250 mM	KCl	18,64 g

Mix the Pipes, CaCl2, and KCl in 900 ml of millipore water. Add NaOH until pH is 6.7,

Don't worry, dust disappear after pH adjust.

Add MnCl2 (see below), stir, adjust volume to 1 L, then filter sterilize. Store at 4C.

Final [c]	Composent	Volume & Mass
55 mM	MnCl2	10,89 g

- Making DMSO Competent Cells : DAY ONE

Grow 12 ml overnight culture of favorite strain of E. coli in 2XTY at 37°C and 250 rpm (We use Denis’ DH5-alpha).

04/06/2015

- Making DMSO Competent Cells : DAY TWO

Inoculate 1 L SOB with 12 ml overnight culture.

Keep 5mL of SOB for initial OD.

Grow culture at 18C and 180 rpm (this temperature is really important as we see a 10-fold decrease in competency when we grow them at room temperature).

- Solution stock Chloramphenicol 34 mg.mL-1 (29,5 mL) :

Weigh out 1 g chloramphenicol and dissolve in 25 ml of 100% ethanol.

Make up volume to 29.5 ml with 100% ethanol.

Sterilize by filtration.

STORAGE : Aliquots of appropriate volume can be stored at -20 °C

- Solution stock Tetracycline 12,5 mg.mL-1 (80 mL) :

Weigh out 1 g tetracycline and dissolve in 75 ml of 1:1 vol/vol distilled water:ethanol.

Make up volume to 80 ml with 1:1 vol/vol distilled water:ethanol. (40mL ethanol + 40mL dH2O)

Sterilize by filtration.

STORAGE : Aliquots of appropriate volume should be wrapped in aluminium foil and stored at -20 °C

05/06/2015

-LB agar 250mL (x3)

Composent	Volume & Mass
Bactotryptone	2,5 g
NaCl	2,5 g
Yeast Extract	1,25 g
Agar	3 g
H2O	qsp 250 mL
Adjust pH to 7.5 with NaOH and autoclave for 20 minutes
+ Chloramphenicol (34 mg/ml in ethanol)	75 µL / 250 mL medium {10 µg/ml (final)}
+Tetracycline (12,5 mg/ml in 50% ethanol)	250 µL / 250 mL medium {12,5 µg/ml (final)}

-LB liquid 500mL

Composent	Volume & Mass
Bactotryptone	5 g
NaCl	5 g
Yeast Extract	2,5 g
H2O	qsp 250 mL
Adjust pH to 7.5 with NaOH and autoclave for 20 minutes

- Making DMSO Competent Cells : DAY THREE

Grow cells until A600 0.5-0.7

Blanck = SOB

A600 nm = 0,011

A600 nm = 0,008

=> So we incubate all week-end at 18°C and 120 rpm.