• True NO BACKGROUND cloning
• Eliminate the colony screening step - all colonies are positive
• Works with any strain of E. coli
• No need for dephosphorylation or complete digestion of vector
• One hour, one step

Figure 1: Map of the CloneSure™ pGATA Positive Selection Vector

Simplify DNA Cloning

The CloneSure™ pGATA positive selection vector is a 5329 base pair general cloning vector which contains a gene coding for a toxic peptide that inhibits bacterial growth when expression is induced (Trudel, P. et al.(1996) BioTechniques 20:684-693). Insertion of a DNA fragment into the multiple cloning site disrupts the gene coding for the toxic peptide, thus rendering the recombinants non-toxic. The vector, in the absence of the insert, produces a toxic peptide which inhibits bacterial replication. Therefore, only colonies which harbor a plasmid containing your cloned insert will grow. This constitutes a background-free positive selection system for recombinant molecules.

The toxic peptide in CloneSure™, which represents a region of the eukaryotic transcription factor GATA-1, binds to the bacterial origin of replication under the control of the IPTG-inducible tac promoter. To prevent leaky expression of the toxic peptide in the absence of IPTG, the CloneSure™ vector also contains the lacI^q repressor gene. CloneSure™ contains the ampicillin resistance gene and a high copy number origin of replication (colEI type). Unlike other similar vectors which require special strains of E. coli to accomplish background-free cloning, CloneSure™ can be used effectively with any strain of E. coli.

Advantages

Use of the CloneSure™ vector system eliminates time consuming steps required in standard cloning protocols. Most standard cloning protocols require that the vector termini be dephosphorylated after digestion to prevent re-ligation of the vector. Many protocols also require the vector be completely digested by using a large excess of restriction enzymes and/or by purifying the digested form of the vector. These steps are not only time-consuming, but also reduce cloning efficiency. Furthermore, to have sufficient vector DNA to perform all ligation experiments and controls, it is common practice to start with 1 to 5 µg of vector DNA. It is therefore difficult to clone DNA fragments available in limited quantities.

The use of CloneSure™ positive selection system eliminates the worry about the quality of your vector preparation and the quantity of vector available. DNA from standard, even "dirty" mini-preps can be used directly without further purification because partial digestion is not a problem. Since the only manipulation is to inactivate the restriction enzyme used to cut the vector, loss of starting material is reduced to a minimum, and a cloning experiment can be completed with less than 1 µg of starting material.

Figure 2: Xba I fragment was cloned into a cloning site of CloneSure™ vector (insert/vector ratio: 1/1). Lane 1, CloneSure™ vector linearized using Xba I. 17 of the 18 (94%) colonies analyzed contain the cloned Xba I fragment.

Applications & Use

Whatever the cloning application is, the CloneSure™ positive selection vector will screen the right clones for you without radioactivity. From the routine cloning of DNA using positive selection to the construction of cDNA libraries, it works for you. In sequencing, the selection system allows directional cloning using the versatility of the Multiple Cloning Site (MCS) without the need to purify the vector or the recombinant fragments. The Amp^r/GATA selection ensures the growth of only your recombinants with the additional flexibility of using a variety of common E. coli strains.

Typically, after ligation of 100 ng of CloneSure™ with an insert:vector ratio of 2:1 in a 10 µl reaction volume, transformation of DH5 competent cells (efficiency of 3 x 10⁷ transformants/µg DNA) yields more than 10⁴ colonies and greater than 90% recombinants. CloneSure™ is supplied at 0.5 µg/µl and each vial contains enough vector for 100 ligations.