Exam 2 Flashcards
(23 cards)
What is the purpose of PCR?
Amplify certain regions of DNA Into copies
What are the basic steps of PCR and what occurs in each one?
- Denature → denaturation of template into single strands
- Anneal → annealing of primers to each original strand for new strand synthesis
- DNA Synthesis → extension of the new DNA strands from the primers
What are the general steps of bacterial transformation?
- Cell Prep → mix bacteria with DNA
- Cell Transformation → heat shock bacteria
- Cell Recovery → add media to cells
- Cell Plating → Bacteria plated on media containing an antibiotic
Why do we heat shock bacteria?
it increases membrane permeability to DNA Molecules
How do you undo heat shock (i.e. close the membrane back up so nothing leaks out)?
Ice
What is the purpose of plating bacteria with antibiotics?
The antibiotic on the plate helps select the bacteria that have taken up the plasmid
How do you validate that the selected bacteria did take up the plasmid?
tests like Restriction Digest and PCR
What is the purpose of bacterial transformation?
Transform/Use bacteria to produce multiple copies of a DNA molecule
What are the basic steps in plasmid isolation (mini-prep protocol)?
- Resuspend, Lysis, Neutralize
- Bind DNA
- Wash the Column
- Elute purified DNA
What are the basic steps of Restriction Digest?
- Isolate plasmid from bacteria
- Digest plasmids with restriction enzymes
- Resolve digest by agarose gel and determine banding patterns
How do you predict the expected results of a Restriction Digest?
- Verify total plasmid size
- Choose enzyme that cuts at least once in the insert (gene) and one that cuts outside the gene in the vector
- Determine where primer will cut and into how many pieces
- Find the size of each piece to get band sizes
- Subtract the number where the piece stops from where it started
- If what you calculated is what appears in the PCR, you calculated correctly
What are the basic steps of the TriReagent protocol?
- Wash cells
- Scrape cells
- Centrifuge → remove supernatant
- Add trizol → incubate
- Add chloroform → shake → incubate → shake
- Centrifuge
- Add isopropanol to clean tube and add in aqueous phase → incubate
- Centrifuge → remove supernatant
- Wash
- Centrifuge → remove supernatant
- Resuspend
- Analyze on spec
What are the basic steps of transfection?
- Add JetPrime to DNA
- Incubate
- Add complex to cells
- Assay cells for 48 hours
In the Plasmid DNA Mini Prep, why should you not vortex the tube and incubate for more than 5 minutes after the lysis step?
Vortexing can shear the chromosomal DNA
incubating for more than 5 minutes can cause denaturation of supercoiled plasmid DNA
In the Plasmid DNA Mini Prep, why is it important to mix thoroughly after the neutralization step?
to avoid localized precipitation of bacterial cell debris
In the Plasmid DNA Mini Prep, why is the wash step important?
to remove trace nuclease activity
In the Plasmid DNA Mini Prep, why is it important to centrifuge the pellet again after discarding the flow through of the final wash?
to avoid residual ethanol in plasmid preps
Why is ethanol used instead of isopropanol?
ethanol is more volatile than isopropanol so it evaporates quicker
isopropanol → Ethanol → Methanol
rpm
revolutions per minute
based on diameter of rotor
g
centripetal force (RCF)
g-force → gravity
In RNA Isolation from Cells, after the cells and all the reagents are added to the tube and centrifuged, what occurs in the tube?
Three phases should be apparent: a lower red, phenol-chloroform phase, a white interphase, a colorless, upper, aqueous phase
Which phase is RNA located in?
exclusively in the upper aqueous phase
What things might appear in the middle interface phase?
protein, DNA, lipids, and carbohydrates
