Ayoung investigator would like to express and purify a newly discovered gene. she decides to clone the gene into a plasmid that contains the sequence for a hexahistidine tag. plasmids that contain a sequence encoding a hexahistidine tag will produce a recombinant protein with an additional six histidine residues attached to the n (or c) terminus. her gene of interest will be expressed as an n-terminal fusion protein containing the six residue histidine tag (n- his6-protein -c). order the steps for construction of the expression plasmid. (note: the gene of interest will be inserted between the noti and hindiii sites.)
step 1:
step 2:
step 3:
step 4:
step 5:
the options are:
-introduce notl and hindlll restriction sites at the ends of the gene of interest via pcr.
-digest the plasmid and dna encoding gene with the notl and hindlll restriction endonucleuses
-transform the plasmid into e. coli for expression
-purify the protein using a column of immobilized ni^2+
-ligate the cut plasmid and dna fragment
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Ayoung investigator would like to express and purify a newly discovered gene. she decides to clone t...
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