molecular bio lab final (copy)

genes in a bottle lab
cell source

cheek epithelial cells

genes in a bottle lab
lysis buffer purpose

to break down cell and nuclear membranes

genes in a bottle lab
protease purpose

remove any proteins associated with the dna
inactivate enzymes that may break down dna

genes in a bottle lab
sodium chloride purpose

neutralize negative charges on dna, allowing strands to stick together

genes in a bottle lab
alcohol purpose

precipitates dna out of solution to make it visible

genes in a bottle lab
appearance of dna in tube at the end

fine white strands

care/use of microscope
cleaning lenses

only use lens paper

care/use of microscope
objective and ocular lenses

objective lenes sit under ocular (4, 10, 40, 100)

care/use of microscope
coarse and fine focus

course: 4 and 10
fine: 40 and 100

care/use of microscope
oil immersion

only with 100, enhances resolution

restriction enzyme lab
what are restriction enzymes?

biomolecules that are able to cut or cleave dna

restriction enzyme lab
what are restriction enzymes isolated from?

different types of bacteria

restriction enzyme lab
restriction sites

dna sequences that the enzymes recognize

restriction enzyme lab
what does cutting dna at restriction sites produce?

different sized dna fragments

restriction enzyme lab
samples used in the experiment

L uncut dna
P pst I
E eco rI
H hind III

restriction enzyme lab
loading dye

added to samples to make them visible, does NOT stain dna

restriction enzyme lab
how is agarose gel made?

end caps on either side if cassette need to be raised
well comb

restriction enzyme lab
electrophoresis chamber set up

put gel in chamber
drop plastic endcaps
gel wells toward negative end

restriction enzyme lab
buffer purpose

helps to conduct electrical current for electrophoresis

restriction enzyme lab
staining gels: ethidium bromide

binds to the dna between base pairs
STAINS DNA

restriction enzyme lab
dna fragment migration: size

larger near top of gel, smaller near bottom

restriction enzyme lab
nature of agarose

porous, dna runs through pores

pGLO lab - bacterial transformation
construct of pGLO plasmid

GFP (green florescent protein)
Bla gene (antibiotic resistance)
Ara c (binding protein that regulates GFP expression)

pGLO lab - bacterial transformation
bacterial cells

E coli

pGLO lab - bacterial transformation
process of transforming E coli

CaCl
heat shock

pGLO lab - bacterial transformation
experimental results

growth and glowing bacteria

pGLO lab - bacterial transformation
arabinose

necessary for expression of GFP gene

pGLO lab - GFP purification
where is fluorescence?

1st centrifuge: pellet
2nd centrifuge: supernatant

pGLO lab - GFP purification
GFP isolation

column chromatography

pGLO lab - GFP purification
lyse cells

lysosome: break down bacterial wall

pGLO lab - GFP purification
why did we freeze the sample?

to put additional stress on the cells, break down bacterial wall

pGLO lab - GFP purification
equilibration buffer

prepares column to receive protein sample

pGLO lab - GFP purification
binding buffer

allows sample to bind to column

pGLO lab - GFP purification
wash buffer

removes proteins that may have stuck to column

pGLO lab - GFP purification
elution buffer

pulls GFP off of column

pGLO lab - GFP purification
final collection

purified GFP

pGLO lab - protein gel electrophoresis
contents of laemlii buffer

buffer (maintains pH)
glycerol (glues density)
bromoptenol blue (loading dye)

pGLO lab - protein gel electrophoresis
proteins magrate solely based on...?

molecular weight, NOT shape or charge

pGLO lab - protein gel electrophoresis'
SDS

attaches negative charge to all amino acids of protein

pGLO lab - protein gel electrophoresis
DTT

reducing agent, helps break any disulfide bonds

PCR: polymerase chain reaction
source of dna

hair follicle cells

PCR: polymerase chain reaction
purpose

make copies of a target gene for analysis

PCR: polymerase chain reaction
Kary Mullis

biochemist who developed concept of isolating dna and making copies

PCR: polymerase chain reaction
thermal cycler

copies of small amounts of dna sequences are amplified in a series of temperature changes

PCR: polymerase chain reaction
instagene matrix

binds divalent cations [Mg2+] that are enzyme cofactors

PCR: polymerase chain reaction
enzyme cofactor example

DNAases (inactivate enzymes)

PCR: polymerase chain reaction
master mix

contains all additional elements (besides dna) necessary to carry out PCR reaction (dna replication)

PCR: polymerase chain reaction
contents of master mix

dna polymerase (heat stable)
free nucleotides
buffer
primers

care/use of micropipette
1ml to microliters

1ml = 1000 microliters

care/use of micropipette
p10 limits

1.0-10.0 microliters

care/use of micropipette
p20 limits

2.0-20.0 microliters

care/use of micropipette
p200 limits

20-200 microliters

care/use of micropipette
p1000 limits

200-1000 microliters