Lab 2: Restriction Digest and Ligation

Question 1

What is the purpose of having a multiple cloning site in a plasmid?

Answer 1

They are engineered to have many restriction enzyme cut sites, so there are lots of possible enzymes to use

Question 2

Why do we typically use at least 2 restriction enzymes instead of just one?

Answer 2

Creates bias against intramolecular ligation, which would just create the plasmids we started with

Question 3

Which type of restriction enzyme is best for cloning: the ones that leave blunt ends or sticky ends?

Answer 3

Sticky ends, they leave a single strand overhang that can complementary base pair to a piece of DNA we want

Question 4

Which 3 places on a plasmid do we not want to have a restriction enzyme cut?

Answer 4

Ori, the antibiotic resistance gene, or the desired gene

Question 5

What 4 features need to be present on a plasmid when choosing a cloning vector and why?

Answer 5

1. Origin of replication - allows the cell machinery to replicate our plasmid and create tons of copies
2. Multiple cloning site - has lots of restriction enzyme cut sites, so we can use the vector for lots of different projects
3. A selectable marker - usually antibiotic resistance, allows us to screen for the colonies that were transformed
4. LacZ: a selectable marker that allows us to screen for a vector with an insert

Question 6

What features are present in the pGFPuv plasmid we used?

Answer 6

The GFP gene, two restriction enzyme cut sites that encompass the GFP gene, Ori, and the ampicillin resistance gene

Question 7

What features are present in the pHSG298 plasmid we used?

Answer 7

Ori, Kanamycin resistance gene, multiple cloning site, and the lacZ gene

Question 8

What was the purpose of the negative control for the restriction digest?

Answer 8

Ensure that there was no contamination of the plasmids