11. List at least 4 different combinations that can result from the ligation reaction (including your desired recombinant plasmid as possibility "A"). Ligation doesn’t always result in recombinants; in other words, fragments from different plasmids don’t always anneal/ligate...fragments from the same plasmid can anneal too. When deciding on growth (or not) ask yourself if there is an ori.

A) Sticky-end ligation: It's when in the cloning experiments the most commonly restriction enzymes generate a 4-base single-stranded overhang called the "sticky" or "cohesive" end. these "sticky" ends anneal to other compatible cohesive ends. Then this two ends form a one single "cohesive" or "sticky" ligation.

This commonly used method is performed at a temperature of 12-16°C, or at room temperature, or alternatively at 4°C for a longer period.

B) Blunt-end ligation: This method does not requires a base-pairing of the protruding ends. That is why any blunt end may be ligated to another blunt end. The restriction enzymes SmaI and EcoRV are commonly use to generate the "Blunt-ends". The biggest benefit of using this method is that the desired insert does not require any restriction sites in its sequence, because the blunt-ends are usually generated on a normal PCR. However, the "sticky-end" ligation is more efficient, since the blunt-end method is 100 times slower. That is because it does not have protruding ends, so the ligation reaction depends on random collisions between the blunt-ends and therefore is consequently much less efficient. So in order to compensate for the lower efficiency; the concentration of ligase used is 10 times higher than sticky end ligation (10x or more)

C) Topoisomerase-mediated ligation; This method is used to avoid applaying ligase for ligation, and the cloning process is more faster because there is no need to use a restriction digest of the vector or insert. In this method an attached topoisomerase I activates a linearized vector, then the "TOPO-activated" vector will accept a PCR product by ligating to both of the 5' ends of the PCR product, the topoisomerase is released and a circular vector is formed in the process.

D) Homologous recombination; This is also a method that does not requires to use ligase, thanks to the use of a DNA recombination process. One example is the "Gateway cloning system", in which The gene cloned into the cloning vector, may be introduced by recombination, into a variety of expression vectors.