Lab 5: PCR analysis and Diagnostic Digests

Question 1

What can a diagnostic digest tell us?

Answer 1

The size of the insert, the orientation of the insert, identifying the backbone plasmid and identifying the insert

Question 2

What are 4 criteria for choosing the best restriction enzymes for a diagnostic digest?

Answer 2

• Produces 2-4 fragments
• Produces fragments that can be resolved on a gel (not too big and not too small)
• Reasonably cheap and don’t require special conditions
• Cut at least once in the insert and once in the backbone

Question 3

What were the 4 samples subjected to the diagnostic digest?

Answer 3

Minipreped white fluorescent colony, minipreped white non-fluorescent colony, pGFPuv and pHSG298

Question 4

What was the purpose of digesting the pGFPuv and pHSG298 plasmids during the diagnostic digests?

Answer 4

Negative controls, what we should see if there was no recombinant plasmid