Genetics Lab Practical 1

Front 1

PCR components

Back 1

DNA template, primers, taq polymerase, dNTPs, buffer, divalent cations, double-distilled water

Front 2

dNTPs

Back 2

Deoxyribonucleotide triphosphates: DNA monomers

Front 3

Taq buffer

Back 3

for stable salt concentration and pH for PCR

Front 4

Divalent cations

Back 4

usually MgCl2, enzyme cofactors required for strand extension by Taq polymerase

Front 5

Thermal cycler temperatures for PCR

Back 5

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

Front 6

agarose is....

Back 6

A polysaccharide purified from agar, which comes from seaweed.
A disacchardie composed of galactose and galactopyranose

Front 7

draw agarose

Back 7

(blank) look up lab 2

Front 8

the agarose matrices serve as ______

Back 8

molecular sieves

Front 9

markers

Back 9

DNA fragments of know lengths

Front 10

how do we see DNA in the gel?

Back 10

ethidium bromide

Front 11

loading dye

Back 11

EDTA, glycerol, tracking dyes

Front 12

EDTA

Back 12

in loading dye, chelates with cation cofactors of DNAases to protect DNA

Front 13

tracking dyes

Back 13

bromophenol blue or xylene cyanol

Front 14

the name of our gene

Back 14

TvRad51

Front 15

the name of our vector

Back 15

pBAD-TOPO

Front 16

Three important features of plasmid vectors

Back 16

MCS, ORI, selectable marker

Front 17

MCS

Back 17

multiple cloning site or polylinker

Front 18

ORI

Back 18

origin of replication, allows plasmid to replicate independently of host chromosomal DNA

Front 19

Selectable marker

Back 19

resistance to specific antibiotic, in our case ampicillin

Front 20

what is special about pBAD-TOPO that makes it awesome for inserting PCR product into?

Back 20

Taq polymerase makes sticky A on 3' ends of PCR product, and the pBAD-TOPO vector has overhanging Ts.

Front 21

what is the purpose of topoisomerase in our ligation?

Back 21

topoisomerase catalyzes formation of new phosphodiester linkages to seal the gaps between the cloning vector and the insert

Front 22

definition of transformation

Back 22

genetic alteration of a cell as a result of the uptake of foreign genetic material

Front 23

how do we make cells chemically competent?

Back 23

soak bacteria in divalent cations such as Ca+2

Front 24

3 stages of transformation

Back 24

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

Front 25

what is the purpose of cation solution in incubation step of transformation?

Back 25

they neutralize phosphates on DNA and phospholipids of cell membrane to allow DNA to get to the cell membrane

Front 26

disadvantages of electroporation

Back 26

requires more cells and special instrumentation (more efficient though)

Front 27

agarose gel is made of ______

Back 27

1% agarose, running buffer (0.5X TBE), and ethidium bromide

Front 28

TBE = running buffer

Back 28

Tris buffer (for slightly basic soln)
Boric acid gives ions for current
EDTA chelates divalent cation cofactors of DNAases

Front 29

the color of the positive pole is _____

Back 29

red

Front 30

components mixed for TOPO ligation

Back 30

PCR product, salt solution, and pBAD-TOPO vector

Front 31

how does topoisomerase come in the pBAD-TOPO vector?

Back 31

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

Front 32

Buffer P1

Back 32

Tris-HCl, EDTA, RNase, lysozyme (digests cell wall)

Front 33

Buffer P2

Back 33

alkaline lysis solution:
SDS (disrupts membranes and denatures macromolecules)
NaOH also denatures macromolecules
(increased viscosity of solution)

Front 34

Buffer N3 (key step)

Back 34

potassium acetate, pH 5.5
-renatures macromolecules non-specifically
-high salts help DNA bind to silica membrane

Front 35

silica matrix

Back 35

add supernatant from previous miniprep steps so only plasmid DNA is added and binds to column

Front 36

Buffer PE

Back 36

ethanol to remove salts from the spin column

Front 37

Buffer EB

Back 37

pH 8.5, elutes DNA with low salt concentration and basic conditions

Front 38

EcoR1 restriction site

Back 38

GAA---

Front 39

EcoRV restriction site

Back 39

GAT---

Front 40

# of base pairs in pBAD vector

Back 40

4 Kb (with EcoRV site about 900 bp before promoter)

4126 (with EcoRV site 950 bp before promoter)

Front 41

# of base pairs in TvRad51 gene

Back 41

990 (with EcoRV site about 100 bp in)

Front 42

what features does pBAD have that make it easy to get proteins out of bacteria?

Back 42

- inducible bacterial promoter
- 6xHis tag
- V5 epitope tag

Front 43

what are the characteristics of ideal primers?

Back 43

- 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

Front 44

How are melting and annealing temperatures related?

Back 44

annealing temperature is about 3˚C below the melting temperature

Front 45

how is melting temperature calculated?

Back 45

4(G+C) + 2(A+T)

Front 46

what are examples of protein tags?

Back 46

6xhis, FLAG (made up)
myc, HA (derived from other proteins)
thioredoxin is a small protein that increases stability of the target protein

Front 47

we purified our protein using a technique called_________
with magnetic beads with ____ ions. ____ ions could also be used.

Back 47

affinity chromatography
nickel
cobalt

Front 48

how do we elute our proteins from the magnetic beads?

Back 48

excess imidazole competes with the 6xHis-tagged protein for nickel, displacing it

Front 49

what can cause problems with purifying protein, and what do we have to do to solve the problem?

Back 49

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

Front 50

Steps of affinity chromatography

Back 50

- 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

Front 51

SDS-PAGE stands for....

Back 51

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Front 52

what are 3 properties of a protein gel that make it more problematic than for DNA?

Back 52

- 2˚, 3˚, 4˚ structures
- already differently charged
- proteins must enter gel at the same time

Front 53

how do we solve the problem of protein 2˚, 3˚ and 4˚ structure in running a gel?

Back 53

- SDS (denatures)
- DTT (dithiothreitol, breaks disulfide bridges)
- boiling (increases rate of denaturation)

Front 54

how do we give the proteins a uniform charge to run through a gel?

Back 54

each SDS molecule associates with about 2 amino acids giving a uniform negative charge proportional to the protein's molecular weight

Front 55

draw an acrylamine monomer

Back 55

H2C=C-CO-NH2

Front 56

polyacrylamide gels are different from agarose gels because....

Back 56

- more resolving power
- vertical
- harder to pour and more expensive
- harder to purify DNA fragments from

Front 57

what happens in a stacking gel?

Back 57

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

Front 58

what happens when proteins enter the resolving gel?

Back 58

- pH of 8 makes glycine negatively charged so it speeds ahead of proteins
- proteins now migrate according to size

Front 59

what is the dye used to visualize all proteins after SDS-PAGE?

Back 59

Coomassie Brilliant Blue to stain the entire gel

Front 60

What helps proteins stick to the nitrocellulose membrane?

Back 60

methanol
(and the membrane's very high affinity for amino acids)

Front 61

what is in the transfer buffer for a Western?

Back 61

methanol to help proteins stick to nitrocellulose membrane

Front 62

what do we do after running the Western transfer for 1 h?

Back 62

submerge the cassette in blocking buffer and incubate at 4˚C overnight

Front 63

antigen stands for _______ and the parts of antigens where antibodies bind are called ______

Back 63

antibody generator
epitopes

Front 64

where do we get primary antibodies from? Which animal is ours from?

Back 64

rabbits, mice, goats, sheep, or horses
RABBITS

Front 65

what ways can we label secondary antibodies?

Back 65

fluorescence
radioactivity
enzyme like HRP or alkaline phosphatase

Front 66

what is the name of our secondary antibody?

Back 66

goat Anti-Rabbit

Front 67

how do you see AP-labeled antibodies?

Back 67

Western Blue substrate contains BCIP and NBT that alkaline phosphatase turns purple

Front 68

What solution is the primary antibody in when we add it to our nitrocellulose membrane to do a Western blot?

Back 68

TBST: 0.1% Tween-20 in 1x Tris buffered saline solution
(or 5 mL Blocking buffer = 5% milk in TBST)

Front 69

What are the steps to Western blotting after the nitrocellulose membrane has our protein on it?

Back 69

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

Front 70

What is Tween-20?

Back 70

A detergent that interferes with non-specific binding of antibodies to random proteins or to the membrane itself

Front 71

What does TBST contain?

Back 71

TBS (Tris buffered saline soln)
Tween-20