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Research Diagnostic Therapeutic

The StabyCloning™ kit is an easy-to-use kit designed for the rapid and efficient cloning of blunt end PCR products. At the end of the fast cloning procedure (less than 1 hour including plating), while background is nearly zero, the PCR product is cloned into one single orientation and is perfectly stable. Finally the stabilized insert is easily exportable to any other vector.

SPEED: Ligation + transformation + plating in one hour
PRECISION: Vectors without insert are eliminated (background < 1 %) and the DNA fragment is correctly oriented in the vector
MOBILITY: Export of [stabilization cassette + insert] is easily selected into other vectors
Recombinants are all independent clones (direct plating)
No “satellite” colonies observed even after extended incubation
No risk of background when using AmpR plasmids as PCR template
Recombinant plasmids are perfectly stabilized without the use of antibiotics
The system is usable in any culture medium

Name	Quantity	Reference	EUR
StabyCloning™ kit with cloning bacteria CYS21, electro-competent cells	10 RXNs	GE-STC1-10	210.00
StabyCloning™ kit with cloning bacteria CYS21, chemically-competent cells	10 RXNs	GE-STC1-12	210.00
StabyCloning™ kit with cloning bacteria CYS21, electro-competent cells	20 RXNs	GE-STC1-20	399.00
StabyCloning™ kit with cloning bacteria CYS21, chemically-competent cells	20 RXNs	GE-STC1-22	399.00
Set of cloning bacteria CYS21, electro-competent cells (20 x 50 µl)	20 RXNs	GE-STCB-20	279.00
Set of cloning bacteria CYS21, chemically-competent cells (20 x 100 µl)	20 RXNs	GE-STCB-22	279.00

Delivery times

2 working days

Shipping conditions

Dry ice

Storage conditions

-80 °C

The StabyCloning™ kit contains linearized plasmid DNA of the pSTC1.3 vector, competent (chemically or electro) bacteria, ligase, regeneration medium, sequencing primers, positive control, and the instruction manual. Additional competent bacteria can be purchased separately.

Principle

The CYS21 bacteria strain, included in the StabyCloning™ kit, contains a natural bacterial poison gene (encoding the poison protein ccdB) into its chromosome. A truncated and inactive antidote gene (ccdA) is present in the plasmid vector (pSTC1.2). This vector is linearized at the end of the truncated antidote gene. The ends of the vector are blunt. When a sequence of 14 base pairs (encoding the last

4 codons and the stop codon of the antidote gene) is added to the

5’-end of the DNA fragment to be cloned, the fusion of this sequence with the truncated gene restores an active antidote gene encoding an active protein (CcdA) able to counteract the action of the poison. The 14-bp sequence is incorporated to the DNA fragment using one modified PCR primer. As illustrated below, this process allows the selection of (i) the recombinant plasmids that incorporate the fragment of interest, and (ii) the orientation of the fragment of interest (only one of the two possible orientations will restore an active, non-truncated, ccdA gene).

Results

With the StabyCloning™ kit, the complete cloning procedure is performed in 1 hour including plating. All the plated colonies are independent clones because no growth phase is required between transformation and plating. All growing bacteria contain the cloning vector with an insert in the correct orientation (figure below).

Restoration of an active ccdA gene allows the selection of recombinant bacteria and also stabilizes the plasmid into the bacterial population: if some bacteria lose the vector, they will die due to the action of the bacterial poison. After cloning of an insert, it is easy to export the stabilization cassette (antidote gene) together with the insert using the restriction sites in the vector. The insertion into another vector can be selected using the CYS21 bacteria and the antidote properties.

Legal notices

The StabyCloning™ kit is covered by worldwide patents. The kit is sold under a license from the
Université Libre de Bruxelles (Belgium). The kit is sold for research purpose only. A license
from Delphi Genetics SA is required for any commercial use (Please, contact Delphi Genetics SA
at delphigenetics@delphigenetics.com).