Abstract: . . .  Make a diagram for each of the plasmid molecules, 1 and 2 based on the restriction patterns shown in the gel above.                                        e) Make a diagram of what the plasmid would look like if the DsRed gene fragment had been inserted in the opposite orientation.   Also, make a diagram of what the gel would look like if the HindIII and MfeI/NdeI digests of this plasmid were separated by electrophoresis.                                                                                    f) You would like to generate a single product pDsRedN1 with the DsRed gene fragment in a unique orientation. Use any of the restriction enzymes provided to design such a procedure.                                   Solutions to Recombinant DNA/ Cloning   a)  pN1 would give a single band at 3900 bp and the DsRed gene fragment would give a single band at 800 bp.   b) When you mix the DNA and bacteria in the transformation protocol, most of the bacteria in the transformation reaction do not take up a plasmid. The presence of kanamycin prevents all these other bacteria from growing and allows you to select only those containing the kanamycin resistance gene.   c) please see the gel in the answer to e.   d)       e)         f) Use HindIII and EcoRI to cut the pN1 plasmid and use HindIII and MfeI to cut the DsRed gene fragment. This procedure generates two different sets of compatible "sticky" ends on the vector and insert.  The HindIII overhangs of the vector and the fragment will hybridize and can be ligated together.  The EcoRI overhang on the vector is compatible with the MfeI overhang on the fragment and these will hybridize and can be ligated.  This allows the DsRed gene to insert in only one orientation.    . . .
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