LABORATORY 4A:
Making Sure You’ve Got A
Recombinant Plasmid
2014
Plasmid of interest, pARA-R
Restriction digest of pARA-R
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
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
DL
R–
R+
Multimer
Nicked
Supercoiled
5 Kb
Linear Fragment
Linear Fragment