Team:IONIS Paris/Notebook

Notebook

• Mar.
  • 01.04.15
  • 30.03.15
  • 28.03.15
• Apr.
  • 01.04.15
• May
  • 29.05.15
  • 28.05.15
  • 26.05.15
  • 25.05.15
  • 21.05.15
  • 13.05.15
  • 07.05.15
  • 06.05.15
• June
  • 10.06.15
  • 09.06.15
  • 05.06.15
  • 04.06.15
  • 02.06.15
• July
• Aug.
• Sept.

Results

Results from the liquid cultures: all the cultures have grown

Miniprep

Aim: plasmid purification from transformed bacteria

Protocol from MO BIO, UltraClean® Standard
Mini plasmid Prep Kit l
4 plates with Ampicillin

Dilution of the glycerol in water
Percentage in mass not in volume
Ex: 50% glycerol solution 50 g Glycerol + 50 g water (mQ)
Then filtration with vacuum pump

Glycerol preparation & Glycerol Stock

Aim: store the transformed bacteria for later use

Weight 50 g of pure Glycerol
Weight 50 g of distilled water
Mix and homogenize the solution
Filtration with vacuum pump, then close the bottle under PSM
Under PSM class II: put 1ml of liquid culture in a 1,5 ml sterile tube (pDusk, pDawn, psB1C3, VVD)
Centrifugate 1mL of liquid culture 4000 rpm, 5 min
Remove and discard the supernatant
Under PSM: resuspend with 0,5 ml of LB without antibiotics
Add 0,5 ml of glycerol 50% (25% final)
Homogenize
Transfer in an identified cryotube and store at -80°C

1 April 15

Results

Results observation for the transformed cells culture (in Petri dishes): no colonies for the positive control pUC19, other plates and negative controls are as expected.

Liquid culture

Antibiotics concentration:
Use of 3 colonies (3 tubes) from VVD, pDawn, pDusk, pSB1C3 3ml LB Broth + 3µl
Antibiotic in double click tubes (12ml) 37°C, 120rpm, O/N

31 March 15

Amplification of pDusk, pDawn, pSB1C3, VVD, pUC19 into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 50 ml of LB agar into a Falcon tube (50ml) + 50 µl antibiotic
For 25 ml / plate (2 plates)

Agar plates preparation

Plasmid	Antibiotic	Number of plate	Remarks
pDusk
	Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pDawn
	Kanamycin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pSB1C3
	Chloramphenicol	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
VVD
	Amplicilin	1	50 µl of transformed cells
		1	100 µl of transformed cells
		1	50 µl of competent cells (neg control)
pUC19	Amplicilin	1	50 µl of transformed cells

Summary:
6 plates with Kanamycin
3 plates with Chloramphenicol
4 plates with Ampicillin

Antibiotics concentration:
Ampicillin: 50 µg.ml^-1 diluted 1/1000
Chloramphenicol: 25 µg.ml^-1 diluted 1/1000
Kanamycin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation

Into aliquots, added plasmids or nothing (control) as following:

Tube n°	Reagent added	Volume (µl)			Tube n°	Reagent added	Volume (µl)
1	pDusk	1			5	pSB1C3	1
2	pDawn	1			6	(Amp control)	/
3	VVD	1			7	(Kan control)	/
4	pUC19	1			8	(Cam control)	/

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the 3 control plates
Many colonies into all the other plates;
Colonies from pSB1C3 transformation should exhibit red color

30 March 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