Week 8 Flashcards
Blue/White screening
Lyse plasmid, creating sticky ends. Blue has disruption, whilst white has gene of interest.
LUC reporter
Uses firefly’s luciferase and measure decay. Needs strong microscope and is often used for animals.
GFP reporter
Light activated and often hybridises with protein of interest. But can move around, so location is imprecise
Forster resonance energy transfer
Donor( often gfp) responds to light signal, transfer to acceptor, energy release dependent on space can be detected.
Phage display technology
Clone gene of interest into coat of bacteriophage, allowing for external protein exposure. Assay against these immobilised proteins will lead to nonbinding phage.
DNA centered assay for protein:DNA interactions (PDI) (yeast 1-hybrid)
Construct1: bait DNA w/ GAL4
Construct2: prey TF(transcriptor factor) if it fuses to DNA —> GAL4 —> AD (reporter) activation
TF(transcriptor factor)-centred assay for protein::dna interaction (eg ChiP)
Make antibody specific to protein, can purify factor from many proteins and can then test bind to DNA
SELEX
Transcriptor factor, interact to cDNA library and hybridise. Scan microarray to find region of best binding
Calcium phosphate co-precipitate method for inserting DNA into mammalian cells?
DNA with calcium phosphate precipitates the DNA, which can then be added to cell and incorporated.
Lipofection
Put DNA into the middle of vesicles, allowing for entry into cell.
Particle bombardment/Gene gun
Physically blast DNA+gold or tungsten into organism. However, random insertion.
De novo pathway for selection characteristics
Put something new that allows for selection. No disruption of native pathways
Salvage pathway for selection characteristics
Use cells unable to do something, transgene has the gene for that.
What is gene amplification
Select for high expression. Either multi-copies or avoiding condensed areas of the DNA.
