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71 Cards in this Set

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PCR components
DNA template, primers, taq polymerase, dNTPs, buffer, divalent cations, double-distilled water
dNTPs
Deoxyribonucleotide triphosphates: DNA monomers
Taq buffer
for stable salt concentration and pH for PCR
Divalent cations
usually MgCl2, enzyme cofactors required for strand extension by Taq polymerase
Thermal cycler temperatures for PCR
94˚ for 3 min
30 to 40 cycles of:
94˚ for 30 sec
56-64˚ for 30 sec
72˚ for 1-3 min
then 72˚ for 5 min
10˚ indefinitely
agarose is....
A polysaccharide purified from agar, which comes from seaweed.
A disacchardie composed of galactose and galactopyranose
draw agarose
(blank) look up lab 2
the agarose matrices serve as ______
molecular sieves
markers
DNA fragments of know lengths
how do we see DNA in the gel?
ethidium bromide
loading dye
EDTA, glycerol, tracking dyes
EDTA
in loading dye, chelates with cation cofactors of DNAases to protect DNA
tracking dyes
bromophenol blue or xylene cyanol
the name of our gene
TvRad51
the name of our vector
pBAD-TOPO
Three important features of plasmid vectors
MCS, ORI, selectable marker
MCS
multiple cloning site or polylinker
ORI
origin of replication, allows plasmid to replicate independently of host chromosomal DNA
Selectable marker
resistance to specific antibiotic, in our case ampicillin
what is special about pBAD-TOPO that makes it awesome for inserting PCR product into?
Taq polymerase makes sticky A on 3' ends of PCR product, and the pBAD-TOPO vector has overhanging Ts.
what is the purpose of topoisomerase in our ligation?
topoisomerase catalyzes formation of new phosphodiester linkages to seal the gaps between the cloning vector and the insert
definition of transformation
genetic alteration of a cell as a result of the uptake of foreign genetic material
how do we make cells chemically competent?
soak bacteria in divalent cations such as Ca+2
3 stages of transformation
Incubation: DNA added to comp cells (in cation solution), at 0˚C
Heat shock: 42˚C, then returned to 0˚C: creates thermal imbalance to create a draft that sweeps plasmid into the cell
Recovery: LB broth, incubation at 37˚C, time to recover and express resistance markers
what is the purpose of cation solution in incubation step of transformation?
they neutralize phosphates on DNA and phospholipids of cell membrane to allow DNA to get to the cell membrane
disadvantages of electroporation
requires more cells and special instrumentation (more efficient though)
agarose gel is made of ______
1% agarose, running buffer (0.5X TBE), and ethidium bromide
TBE = running buffer
Tris buffer (for slightly basic soln)
Boric acid gives ions for current
EDTA chelates divalent cation cofactors of DNAases
the color of the positive pole is _____
red
components mixed for TOPO ligation
PCR product, salt solution, and pBAD-TOPO vector
how does topoisomerase come in the pBAD-TOPO vector?
topoisomerase is attached at each end of the linear vector to link PCR product with 3' sticky As to the vector's 3' sticky Ts
Buffer P1
Tris-HCl, EDTA, RNase, lysozyme (digests cell wall)
Buffer P2
alkaline lysis solution:
SDS (disrupts membranes and denatures macromolecules)
NaOH also denatures macromolecules
(increased viscosity of solution)
Buffer N3 (key step)
potassium acetate, pH 5.5
-renatures macromolecules non-specifically
-high salts help DNA bind to silica membrane
silica matrix
add supernatant from previous miniprep steps so only plasmid DNA is added and binds to column
Buffer PE
ethanol to remove salts from the spin column
Buffer EB
pH 8.5, elutes DNA with low salt concentration and basic conditions
EcoR1 restriction site
GAA---
EcoRV restriction site
GAT---
# of base pairs in pBAD vector
4 Kb (with EcoRV site about 900 bp before promoter)

4126 (with EcoRV site 950 bp before promoter)
# of base pairs in TvRad51 gene
990 (with EcoRV site about 100 bp in)
what features does pBAD have that make it easy to get proteins out of bacteria?
- inducible bacterial promoter
- 6xHis tag
- V5 epitope tag
what are the characteristics of ideal primers?
- 20-25 bp
- 50-60% G-C
- 3' ends not complementary
- forward and reverse have same annealing temperatures
- no tandem repeats
- no long stretches of same nucleotide
How are melting and annealing temperatures related?
annealing temperature is about 3˚C below the melting temperature
how is melting temperature calculated?
4(G+C) + 2(A+T)
what are examples of protein tags?
6xhis, FLAG (made up)
myc, HA (derived from other proteins)
thioredoxin is a small protein that increases stability of the target protein
we purified our protein using a technique called_________
with magnetic beads with ____ ions. ____ ions could also be used.
affinity chromatography
nickel
cobalt
how do we elute our proteins from the magnetic beads?
excess imidazole competes with the 6xHis-tagged protein for nickel, displacing it
what can cause problems with purifying protein, and what do we have to do to solve the problem?
If the protein is unhappy in bacteria or made as such high levels that it clumps together, then you have to exchange the native condition buffer for denaturing conditions
Steps of affinity chromatography
- cell lysis reagent (buffer and detergents)
- DNase I (removes viscosity)
- MagneHis Ni particles
- remove supernatant
- Binding/Wash buffer
- remove supernatant, repeat wash and removal
- Elution buffer (500 mM imidazole)
- save supernatant
SDS-PAGE stands for....
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
what are 3 properties of a protein gel that make it more problematic than for DNA?
- 2˚, 3˚, 4˚ structures
- already differently charged
- proteins must enter gel at the same time
how do we solve the problem of protein 2˚, 3˚ and 4˚ structure in running a gel?
- SDS (denatures)
- DTT (dithiothreitol, breaks disulfide bridges)
- boiling (increases rate of denaturation)
how do we give the proteins a uniform charge to run through a gel?
each SDS molecule associates with about 2 amino acids giving a uniform negative charge proportional to the protein's molecular weight
draw an acrylamine monomer
H2C=C-CO-NH2
polyacrylamide gels are different from agarose gels because....
- more resolving power
- vertical
- harder to pour and more expensive
- harder to purify DNA fragments from
what happens in a stacking gel?
Laemmli's invention:
- gel contains chloride ions
- running buffer has glycine ions
- they create "ion sandwich"
- only 4% acrylamine, so proteins all migrate together regardless of size
- pH 6.8: acidic to keep glycine ions behind proteins
what happens when proteins enter the resolving gel?
- pH of 8 makes glycine negatively charged so it speeds ahead of proteins
- proteins now migrate according to size
what is the dye used to visualize all proteins after SDS-PAGE?
Coomassie Brilliant Blue to stain the entire gel
What helps proteins stick to the nitrocellulose membrane?
methanol
(and the membrane's very high affinity for amino acids)
what is in the transfer buffer for a Western?
methanol to help proteins stick to nitrocellulose membrane
what do we do after running the Western transfer for 1 h?
submerge the cassette in blocking buffer and incubate at 4˚C overnight
antigen stands for _______ and the parts of antigens where antibodies bind are called ______
antibody generator
epitopes
where do we get primary antibodies from? Which animal is ours from?
rabbits, mice, goats, sheep, or horses
RABBITS
what ways can we label secondary antibodies?
fluorescence
radioactivity
enzyme like HRP or alkaline phosphatase
what is the name of our secondary antibody?
goat Anti-Rabbit
how do you see AP-labeled antibodies?
Western Blue substrate contains BCIP and NBT that alkaline phosphatase turns purple
What solution is the primary antibody in when we add it to our nitrocellulose membrane to do a Western blot?
TBST: 0.1% Tween-20 in 1x Tris buffered saline solution
(or 5 mL Blocking buffer = 5% milk in TBST)
What are the steps to Western blotting after the nitrocellulose membrane has our protein on it?
Blocking solution (buffer + 5% powdered milk)
1˚ antibody in TBST (60 min)
wash with TBST x 3
2˚ antibody in TBST (60 min)
wash with TBST x 3
wash with TBS to remove Tween-20
Western Blue substrate for AP
What is Tween-20?
A detergent that interferes with non-specific binding of antibodies to random proteins or to the membrane itself
What does TBST contain?
TBS (Tris buffered saline soln)
Tween-20