Lecture #3 - DNA Transformation & Plasmid DNA Purification (Mini-Prep)

Question 1

Plasmid

Answer 1

Self-replicating extrachromosomal DNA molecules in bacteria
Contain unique genes for antibiotic resistance not found in the chromosome
Smaller than chromosome; easy to purify

Question 2

What are the three components of artificially constructed vectors? Which one is necessary for maintaining the presence of the plasmid in the cell? (QUIZ #1)

Answer 2

1. Origin or replication aka ori aka replicator
2. Selectable marker i.e. antibiotic resistance
3. Polylinker kaa multiple cloning site

Selectable markers are necessary for maintaining the presence of the plasmid in the cell.

Question 3

What are the two steps to cloning plasmid DNA?

Answer 3

1. Transform in your plasmid DNA into bacteria

2. Mini-Prep

Question 4

Mini-Prep

Answer 4

Kit that is used for purifying plasmid DNA

Question 5

How can you check that your transformed bacteria have the plasmid?

Answer 5

Incubate transformed bacteria on media with the antibiotic the plasmid has resistance to. The ones that grow have the plasmid.

Question 6

Competent bacteria

Answer 6

Bacteria that are able to take up foreign DNA
Bacteria in a competent state
Enable high-effiicienct transformations

Question 7

What are the two steps to making competent bacterial cells for transformation?

Answer 7

1. CaCl2 is added to cells to make them competent by allowing the plasmid to attach to the cell membrane
2. Cells are heat shocked at 42C for 2 minutes, opening the pores of the cell membrane to allow the plasmid to enter the cell

Question 8

Why must we incubate the newly transformed cells for 1 hour at 37C before plating the bacteria on LB/AMP media?

Answer 8

The ampicillin resistance gene first becomes mRNA and then protein, so it needs time for this to occur. Without the protein the cell doesn’t have amp resistance and the cell will die on LB/AMP media.

Question 9

What are the steps for the Mini-Prep?

Answer 9

1. Innoculate a colony into 2 mL of LB/AMP and grow O/N at 37C
2. Spin down O/N culture in 1.5 mL eppendorf tube and discard the supernatant
3. Add both alkaline solution and acid neutralization solutions to the pellet and spin down
4. Run the supernatant through a silica column
5. Wash and elute off with a low salt solution and collect in a tube
6. Check the plasmid DNA to make sure you have isolated the correct one

Question 10

After spinning down the O/N culture in LB/AMP, what is in the supernatant? In the pellet?

Answer 10

LB/AMP in the supernatant and intact bacterial cells in the pellet.

Question 11

Alkaline solution

Answer 11

Alkaline and detergent that lyse the cell
Alkaline denatures both chromosomal and plasmid DNA and makes them ss
Detergent (SDS) dissolves the lipid component of the cell membrane and denatures proteins

Question 12

Acid neutralization solution

Answer 12

Brings pH to neutral (pH=7)

Allows DNA to renature

Question 13

Which renatures first, plasmid DNA or chromosomal DNA and why?

Answer 13

Plasmid DNA because it is smaller

We only want the plasmid DNA to renature

Question 14

What is the role of potassium acetate?

Answer 14

To precipitate SDS along with its lipids and proteins

Question 15

After the acid neutralization step and spinning down the sample, what is in the pellet?

Answer 15

SDS/lipid/protein precipitate traps tangled chromosomal DNA, all of which is in the pellet

Question 16

What is in the supernatant after acid neutralization and spinning down the sample?

Answer 16

Plasmid DNA in a high salt solution

Question 17

How do we get rid of the salt in the solution of plasmid DNA after the acid neutralization step?

Answer 17

Run the supernatant through a silica column, where the plasmid sticks to the silica. Then, wash and elute off with a low salt solution and collect in a tube.

Question 18

The last step in the Mini-Prep is to check the _______ to make sure its the right one.

Answer 18

plasmid DNA

Question 19

Why do we add the sugar arabinose to the plates?

Answer 19

Arabinose is essential for the expression of the GFP gene that is responsible for the green fluorescence used to determine if the plasmid was transferred. Without arabinose, the gene will not express protein and therefore won’t fluoresce under UV light.