● Quick and efficient: Blue/white screening is not necessary, and the whole ligation process can be finished within 5 min.

● Sensitive and widely applied: Good for the ligation of low concentration fragments and long fragments, works for both blunt and sticky end product.

● Without Multiple Cloning Sites (MCS): Suitable for customers who need produce subclones by designing and introducing enzyme digestion site on the vector.

The Lethal Based Simple Fast Cloning Kit (without MCS) is an advanced positive clone selection system for the highest efficiency cloning of PCR products and any other DNA fragment, either blunt or sticky-end. This kit works for both phosphoryl ated and non-phosphorylated DNA fragments. By using this kit, the whole process of positive clone selection and ligation takes only 5 minutes and yields more than 95% positivecl ones. Blunt end PCR products amplified by DNA polymerases which have proofreading activity (e.g., Pfu Polymerase) can be inserted directly into vector. PCR products that amplified by DNA polymerases which don’t have proofreading activity (e.g., Taq Polymerase) need to be treated with Blunting Enzyme in this kit (7 min) before ligation. Ligation products can be used on the transformation of common used E. coli strains.

The kit features a novel positive selection cloning vector pLB-Simpl evector. This vector contains a lethal gene which would be disrupted by the insertion of a DNA fragment into the cloning site. As a result, only cells with recombinant plasmids are able to propagate, cyclized empty pLB-Simple vector will express lethal toxic protein which will kill the E. coli cells.  This cloning method eliminates the need of expensive blue/whitescreening and accelerates the process of cloning and selection.

All components of the Lethal Based Simple Fast Cloning Kit should be stored at -30~-15°C. Avoid repeated freezing and thawing. The shelf life is one year.

● It’s important to have a control experiment during the transformation process, which could help to locate problems.

● We recommend keeping some ligation product for backup in order to avoid unnecessary repetitive experiment.

● Plating amount can be adjusted according to the specific circumstances. For large amount of total DNA, plate less transformation product; for small amount of total DNA, plate 200~300 μl of transformation product. If the predicted number of clones is low, centrifuge the cell culture for 2 min at 4,000 rpm, then remove some of the cell culture any plate the rest. (Keep the rest cell culture at 4°C, plate them at the second day if few clones were produced on the previous plate).