Belton Quiz (OLM)

Question 1

What is bacterial cloning?

What is it used for?

Answer 1

A.K.A. DNA cloning, where bacteria make identical copies of a plasmid

1. Biopharmaceuticals
   
   • Ex., bacteria transcribe and translate an insert to produce insulin
2. Transgenic organisms
3. cDNA libraries
4. Other genome studies

Question 2

What is the ORI?

Answer 2

• Origin of replication
• Replication is initiated here
• Often modified to make it allow more copies of the plasmid in the cell

Question 3

What is the ab^-R?

Answer 3

• Antibiotic resistance gene
• Important because it acts as a selectable marker to ensure only transformed bacteria survive

Question 4

What is the MCS?

Answer 4

• Multiple cloning site
• In the lacZ operon
• Cluster of lots of restriction enzyme cut sites
• Location of DNA of interest insertion

Question 5

What is the importance of the lacZ operon?

Answer 5

• It codes for enzyme β-galactosidase which uses substrate on plate to produce blue pigment
• Colonies with insert are uniquely white because insertion boofs the lacZ site; additional confirmation

Question 6

Outline the cloning protocol

Answer 6

1. Digest plasmid and insert with restriction enzymes
2. Ligate plasmid and insert to combine
3. Transformation occurs (E. coli takes up the recombinant plasmid)
4. Select the colonies with the insert
5. Grow the selected bacteria separately
6. Miniprep bacteria to isolate the plasmidy

Question 7

When digesting, what happens when you use 1 restriction enzyme? 2?

Answer 7

• 1 restriction enzyme allows the insert to enter the plasmid 5’ to 3’ or 3’ to 5’ due to blunt ends
• 2 different restriction enzymes allows the insert to enter the plasmid 5’ to 3’ only

Question 8

What ligase is used?

What is ligation efficieny and what are the 3 outcomes of ligation?

Answer 8

• T4 DNA ligase forms phosphodiester bonds between blunt/sticky ends
• The molar ratio of insert:plasmid is important in determining ligation efficiency
• 3 outcomes:

Question 9

What are competent E. coli?

What are barriers that make it harder to achieve?

Answer 9

• Competent E. coli = treated to easily take up the plasmid
• 2 barriers:
  
  • Negative plasmid, negative inside cell
  • Adhesion zones too small to allow plasmid passage
  • Transformation protocol takes care of this

Question 10

Explain each component of the transformation protocol

Answer 10

Question 11

Describe the miniprep process

Answer 11

• Isolate plasmid from bacteria using spin
  
  1. Lyse bacteria to release plasmid
  2. Add to spin column tube (centrifuge)
  3. Apply wash solutions and centrifuge to dry
  4. Elute plasmid: apply solution that release plasmid from column membrane, and collect
  5. Measure concentration of plasmid using spectrophotometry

Question 12

State the formula for finding the mass of insert (ng)

State the formula for finding the volume of insert (uL)

Answer 12