laboratory 4a: making sure you’ve got a recombinant plasmid 2014
TRANSCRIPT
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
5 Kb
MultimerNicked
Supercoiled
Linear Fragment
R– R+
Linear Fragment
DL