Template:IONIS Paris/Notebook/29-05-15

Liquid culture results

Results of the liquid culture from 28/05/2015

After 24h of growth:
Terminator T7 bacteria growth strongly into both tubes
bJun bacteria growth into strongly one tube only, the other one is “cloudy”
bFos both tube are “cloudy”
Control negative clear

Glycerol stock

Stock of Terminator T7 and bJun (cf protocol for glycerol stock), -80°C

Miniprep

Aim: plasmid purification from transformed bacteria

Protocol from QIAgen, UltraClean® Standard
Mini plasmid Prep Kit
Terminator T7: 2 tubes
bJun: 1 tube

Plasmid digestion (Terminator T7 & bJun)

Terminator T7

Tube	Terminator T7	Control negative
Water	12 µL	13 µL
Buffer 2.1	2 µL	2 µL
DNA	5 µL	5 µL
Enzyme EcoRI	0.5 µL	--
Enzyme SpeI	0.5 µL	--

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• SpeI: 100%

bJun

Tube	bJun	Control negative
Water	12 µL	13 µL
Buffer 2.1	2 µL	2 µL
DNA	5 µL	5 µL
Enzyme EcoRI	0.5 µL	--
Enzyme PstI	0.5 µL	--

NEB double digestion Buffer 2.1:

• EcoRI: 100%
• PstI: 75%

37°C, 1h10
Storage: -20°C

Gel Purification

Aim: purification of fragment from 28.05.2015

Use of a QIAquick gel extraction kit with the protocol of QIAgen
Gel weight = 270 mg

Gibson Assembly

Aim: first step of our VVD Biobrick

Master Mix from University of Freiburg (15µL)
Mix of DNA fragment:

• 1 µL pSB1C3
• 2 µL PCR.2 VVD1
• 2 µL PCR.2 VVD2
• 2 µL YC155

5 µL of the DNA mix is adding to the Master mix
Put immediately into a hot bath at 50°C for 1 hour

Transformation

Aim: transformation of E.Coli with Gibson Assembly product

Transformation

Plasmid	Plate
3 µL of plasmid	20 µL on plate
	50 µL on plate
6 µL of plasmid	20 µL on plate
	50 µL on plate
Control negative	100 µL on plate

Incubate at 25°C during 60 hours

29 May 15