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

2. Plasmid of interest, pARA-R

3. Restriction digest of pARA-R

4. 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

5. 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

6. Running the gel
Wells

7. 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

8. Plasmid configurations
Supercoiled
Nicked circle
Multimer

9. Predict the visualization

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