MMBIO 241 Test Flashcards
What are the four factors that affect enzyme velocity
temperature, enzyme concentration, substrate concentration
Explain the procedure of isolating acid phosphatase.
extract from wheat germ. Smash up wheat germ in extraction buffer, centrifuge, take supernatant which contains the enzyme.
When you want to determin Vmax and Km?
vary substrate concentration, hold time and enzyme concentration constant. graph substrate concentration versus enzyme velocity
to get an accurate value for V0
vary time and hold substrate concentration and enzyme concentration constant. Graph time vs. product concentration
What is happening at Vmax?
enzyme is saturated
what does km represent?
high dm means low affinity. low km means high affinity
whats the purpose of pcr?
amplify a DNA target sequence
What are the three steps in PCR?
denaturation, primer annealing, elongation
Who long is PV92?
300 bp long
What reagents are needed in PCR?
instagene matrix (chelating agent; prevents DNAse from degrading – Protease K), primers, Taq polymerase, PCR buffer, nucleotides
How do you keep your sample from degrading before you run PCR?
ICE ICE BABY
How is Pure Culture used?
single ancestor…. spreading?
How is selective media used?
test against antibiotic
GFP is only activated int he presence of?
Arabinose
What is the formula for transformation efficeincy?
(# colonies)*(dilution factor)/ug plasmid (or DNA)
How do you find dilution factor?
total volume of mixture/volume you spread on plate
Describe how to do GFP chromotography
bacteria suspended in buffer, add lysozyme, incubate on dtry ice, centrifuge, keep supernatent, run supernatent in hydrophobic chromotgraphy
Just the chromotography part
Equilibriation buffer, GFP sample, wash buffer, elution buffer
Steps in our western blot?
run sera from different organisms on native agarose gel, transfer proteins to nitrocellulose membrane, block with gelatin, add antibodies, add color devlopment soultion that causes the enzyme on the antibody to color puprple
darker bands on western blot mean what?
more closely related
how do you know what organisms are more closely related in a phylogenttic tree?
closer together. branch length on the phylogenetic tree does count
