Feb 11 Flashcards
(20 cards)
Do plasmids carry essential genes?
No, often genes for resistance or something, they can be lost/transferred easily
What are the 2 main plasmid uses in lab?
Cloning vectors (store and replicate DNA), and expression vectors (produce RNA and could be the protein product too)
What are 3 plasmid key features needed to use them for expression/cloning?
- origin of replication
- Selectable marker (some way to select bacteria with plasmid like antibiotic resistance
- multiple cloning site, has many unique restriction enzyme sites
What is stringient or relaxed regulation of replication?
Stringent uses host cell’s proteins, relaxed does not
What happens with plasmids with similar ori’s?
They will compete for same replication machinery, so they are incompatible, not grow well together
What controls the plasmid’s copy number?
the Origin of replication (Ori)
What is pos vs neg selection for cells with the plasmid?
Pos is when presence of plasmid allows cell to live EX: antibiotic resistance, or gene required to produce nutrient that is needed for survival.
Neg is when if have plasmid the cell dies.
What is lacZalpha, what is a-complementation?
It is N terminal fragment of lacZ of Bgal. The rest of the AA’s needed for function are laxZM15. They complement eachother to form functional Bgal (alpha-complementation)
What is lactose role in lac operon?
Lactose binds to repressor that would normally prevent lac operon genes transcription, so allows gene transcription now
Bgal is a homo______
tetramer
What is analog of lactose that turns blue when hydrolyzed?
X-gal
How does-Blue white colony screening work?
It is when use plasmid with laxZa, and strain with ZM15. Media has IPTG and X-Gal. IPTG is not metabolized, but does bind to lac repressor so operon turned on. X-gal will be turning blue if plasmid allows both parts of BGAL to come together to be functional
What is phage M13?
It is ssDNA phage, can be used for sequencing,
What conditions to lyse bacterial cells?
SDS and alkaline
How extract plasmids?
After SDS and high pH (denature proteins, chromosomal DNA, but plasmids are supercoiled so unaffected.
Then potassium acetate, potassium dodecyl sulfate is not soluble, and so get rid of all junk, left with plasmids, soluble proteins, ribosomes, RNA in supernatant
How use silica column for plasmid extraction?
High salt makes them bind to column, RNA and proteins don’t bind. Then use low salt to elute plasmids
How do following factors affect DNA migration on agarose gel: Agarose conc, voltage, buffer, dye, DNA conformation?
Agarose conc= more conc. allows better separation of small molecules
Voltage = Lower voltage is slower but better separation
Buffer will affect stuff generally
Dyes are pos, will slow down DNA, make it seem bigger than actual. If all ladder is stained too, then overall cancels out as all affected by dye, could also stain after running gel
DNA conformation: Supercoiled is most compact (appears smaller than actual), then linear, then relaxed circular runs larger than actual
Spectro ratios for DNA contamination?
A260/A230, low ratio indicates contaminating reagents
A260/A280, low ratio indicates proteins or phenol present
What is bioanalyzer?
Get peaks on chromatogram of fluorescence on Y axis, size of DNA on X axis.
