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

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