Making Sure You’ve Got A Recombinant Plasmid LABORATORY 4A: Making Sure You’ve Got A Recombinant Plasmid 2014

Plasmid of interest, pARA-R

Restriction digest of pARA-R

R-tube contains pARA-R plasmid and no restriction enzymes (control) Review of Lab 2A R-tube contains pARA-R plasmid and no restriction enzymes (control) R+ tube contains pARA-R plasmid and restriction enzymes HindIII and BamH I Digested by incubating at 37°C for 60 minutes

Determine if restriction enzymes digested the pARA-R plasmids Goals of Lab 4A Determine if restriction enzymes digested the pARA-R plasmids Use gel electrophoresis to separate the digest products according to “size” Allow visualization of restriction fragments and non-digested plasmids on the gel

Running the gel Wells

What to expect in the visualization Restriction fragments are linear and their speed through the gel is only determined by size Undigested plasmids have multiple configurations and their speed through the gel is determined by size and shape

Plasmid configurations Supercoiled Nicked circle Multimer

Predict the visualization

Results 5 Kb Multimer Nicked Supercoiled Linear Fragment R– R+ DL