Question

PART 3 -SCREENING POTENTIAL CLONES (2 MARKS). After completing a cloning experiment, we screen potential clones to determine which ones are successful. The experiments outlined in Session 2-4 in the lab manual describe how we do this for the LEAPS plasmid. In this experiment using just the EcoRI site to facilitate cloning we have an additional problem - both ends of the PCR product have an EcoRI site and this means that the amplified sequence can be cloned into the PGEX-2T vector in either direction (see diagram below). Eco RI Eco RI Eco RI Eco RI You should describe how you can use a restriction enzyme digest to differentiate between vectors where the insert is in the correct direction and those where the insert is in the incorrect direction You will need to provide: • The names of the restriction enzymes you would use (remember that you can use two In the same digest) The predicted sizes of fragments that would arise from the digestion of a vector with the insert In the correct orientation The predicted sizes of fragments that would arise from the digestion of a vector with the insert in the incorrect orientation • Only a general description of required, you do not need to give recipes of any reactions.