Lecture #3 - DNA Transformation & Plasmid DNA Purification (Mini-Prep) Flashcards
Plasmid
Self-replicating extrachromosomal DNA molecules in bacteria
Contain unique genes for antibiotic resistance not found in the chromosome
Smaller than chromosome; easy to purify
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)
- Origin or replication aka ori aka replicator
- Selectable marker i.e. antibiotic resistance
- Polylinker kaa multiple cloning site
Selectable markers are necessary for maintaining the presence of the plasmid in the cell.
What are the two steps to cloning plasmid DNA?
- Transform in your plasmid DNA into bacteria
2. Mini-Prep
Mini-Prep
Kit that is used for purifying plasmid DNA
How can you check that your transformed bacteria have the plasmid?
Incubate transformed bacteria on media with the antibiotic the plasmid has resistance to. The ones that grow have the plasmid.
Competent bacteria
Bacteria that are able to take up foreign DNA
Bacteria in a competent state
Enable high-effiicienct transformations
What are the two steps to making competent bacterial cells for transformation?
- CaCl2 is added to cells to make them competent by allowing the plasmid to attach to the cell membrane
- Cells are heat shocked at 42C for 2 minutes, opening the pores of the cell membrane to allow the plasmid to enter the cell
Why must we incubate the newly transformed cells for 1 hour at 37C before plating the bacteria on LB/AMP media?
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.
What are the steps for the Mini-Prep?
- Innoculate a colony into 2 mL of LB/AMP and grow O/N at 37C
- Spin down O/N culture in 1.5 mL eppendorf tube and discard the supernatant
- Add both alkaline solution and acid neutralization solutions to the pellet and spin down
- Run the supernatant through a silica column
- Wash and elute off with a low salt solution and collect in a tube
- Check the plasmid DNA to make sure you have isolated the correct one
After spinning down the O/N culture in LB/AMP, what is in the supernatant? In the pellet?
LB/AMP in the supernatant and intact bacterial cells in the pellet.
Alkaline solution
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
Acid neutralization solution
Brings pH to neutral (pH=7)
Allows DNA to renature
Which renatures first, plasmid DNA or chromosomal DNA and why?
Plasmid DNA because it is smaller
We only want the plasmid DNA to renature
What is the role of potassium acetate?
To precipitate SDS along with its lipids and proteins
After the acid neutralization step and spinning down the sample, what is in the pellet?
SDS/lipid/protein precipitate traps tangled chromosomal DNA, all of which is in the pellet