AdEasy™

The AdEasy™ system is used to rapidly generate recombinant adenovirus without the need for time consuming plaque purification. Developed by T.C He et al (12), the AdEasy™ system exploits E. coli's robust, efficient recombination machinery thereby avoiding restriction-ligation involving the unwieldy (36kb) adenovirus genome. Bacterial and 293 cells that are essential to the technique are included in every kit with each kit providing enough reagents to generate up to 5 recombinant adenoviruses. The highly detailed user manual is an invaluable resource for beginners as well as those experienced in this field of work.

STEP 1 : The cDNA of interest is first cloned into a transfer vector.

STEP 2 : The resulting plasmid is linearized with Pme I and co-transformed into E. coli strain BJ5183 together with pAdEasy-1, the viral DNA plasmid. Recombinants are selected with kanamycin and screened by restriction enzyme analysis.

STEP 3 : The recombinant adenoviral construct is then cleaved with Pac I to expose its ITR (Inverted Terminal Repeats) and transfected into QBI-293A cells to produce viral particles.

AdEasy™ Kit

The AdEasy™ kit comes in a complete package format containing all the principal components and controls for the construction of 5 recombinant viruses. Each AdEasy™ system kit includes all the components listed below plus your choice of one transfer vector.

Description of Plasmids and Kit Reagents

Contents of the AdEasy™ Kit

Cat No.	Product	Description	Quantity	Storage
AES1010	pAdEasy-1 ccc DNA plasmid	Ad5E1/E3	0.5µg (100ng/µl; 5µl)	-20°C
AES1005	BJ5183 EC Electrocompetent cells	BJ5183	5 x 80µl	-80°C
AES1007K	DH5 EC Electrocompetent cells	DH5	5 x 40µl	-80°C
AES0503	QBI-293A cells	Frozen 293 cell line	1ml (1 X 106 cells/ml)	-150°C
	QBI-Infect Ad5.CMV-LacZE1/E3	Viral particles of (in complete DMEM)	1ml (›1000 PFU/ml)	-80°C
	CaCl2 2M	Transfection reagent	0.5ml	-20°C
	TE 0.1x	Transfection reagent	0.5ml	-20°C
	HBS 2x	Transfection reagent	3 x 1ml	-20°C
	(see above)	Choice of transfer vector	25µg (500ng/ml; 50µl)	-20°C