Lab Exam 1 Flashcards
(120 cards)
1
Q
week 1 lab goal
A
extract DNA from calf thymus tissue
2
Q
week 2 lab goal
A
quantify extracted DNA (concentration)
3
Q
week 3 lab goal
A
PCR to amplify insulin gene
4
Q
week 4 lab goal
A
ligate insulin gene into plasmid, transform into bacterial cells
5
Q
week 5 lab goal
A
analyze transformants w/ gel
6
Q
3 reasons calf thymus is beneficial
A
- Relatively small cytoplasm
We are only interested in the nucleus, which contains
DNA. We do not want the cytoplasm.
Relatively little protein - Less cytoplasm means fewer proteins like DNase, which
degrades DNA. We want to eliminate DNase so that it
does not degrade the DNA we are trying to extract! - Cheap and easy to get
7
Q
molecular cloning
A
set of experiments where we
use a host organism to create many copies of a gene
* Put human insulin gene into bacteria (like E. coli). The
gene will be amplified as bacteria replicate. We take
advantage of the host’s machinery to produce many
copies of the protein
8
Q
steps of DNA extraction w/ silica membrane
A
9
Q
(lab 1) solution for DNase, enzyme that destroys DNA
A
use EDTA - chelating agent that binds
with cations such as Ca and Mg needed by DNase and forms a water-soluble compound
10
Q
(lab 1) solution to stop other proteins mixing with DNA
A
Proteinase K digests proteins, sodium
dodecyl sulfate (SDS) is a surfactant that lyses cells
11
Q
(lab 1) solution extreme pH environment (DNA is most stable in a neutral pH range)
A
Tris-HCl or PBS - buffer solutions used throughout the experiment to avoid
drastic pH fluctuations
12
Q
proteinase K
A
digests proteins
13
Q
Buffer ATL
A
contains SDS and the chelating agent EDTA
14
Q
SDS
A
detergent used to lyse cells by disrupting non-covalent bonds in protein and denatures them
15
Q
EDTA
A
chelating agent - bonds with Mg and Ca to prevent DNase from getting them
16
Q
Buffer AL
A
lysis buffer - promotes the lysis of the cell membrane, denaturation of proteins and DNase
- contains guanidinium hydrochloride which
promotes DNA binding to the silica membrane
17
Q
Buffer AW1 and AW2
A
wash buffers that keep the DNA bound to the spin column while washing DNA of contaminants and impurities
18
Q
DNA elution buffer
A
water - low in salt, releases DNA from spin column
19
Q
procedure of lab 1
A
- add buffer ATL and proteinase K to calf thymus, vortex and incubate for 1 hour
- add buffer AL and vortex, add ethanol
- Transfer to spin column and
centrifuge to remove lysis
buffer and cell components.
Discard flowthrough - Add AW1 wash buffer. Spin
again. Discard flowthrough - Repeat with AW2 wash buffer
- Spin column without adding
any liquid to remove residual
ethanol - Add water to column and wait
a few minutes. Spin again to
elute DNA
20
Q
optimal temp for proteinase K function
A
56* C
21
Q
1000 microliters
A
1 mL
22
Q
AW1 components
A
guanidine hydrochloride
ethanol
23
Q
AW2 components
A
ethanol
24
Q
guanidine hydrochloride
A
chaotropic salt - high salt content increases DNA binding to silica membrane, + denatures proteins
25
Beer's Law
there is a direct linear relationship between the optical absorbance (A) of a
compound and its concentration
26
A=kc
A = absorbance
k = slope of line of concentration/absorbance graph
c = concentration
27
spectrophotometer
determines absorption of light at a specific wavelength/range of wavelengths
28
k value
50 on standard lab spectrophotometers measuring DNA concentration
29
lab 2 procedure
2-fold (serial) dilutions of DNA with known concentration
1. add 100 µL of 50 ng/µL to tubes 1 and 2
2. add 100µL of TE buffer to tube 2, then continue serial dilution
3. add 100 µL of only TE buffer to tube 5 - will act as a blank
4. prepare unknown 1 - add 90µL of TE buffer and 10 µL unknown DNA sample to tube 6
5. prepare unknown 2 - add 99µL of TE buffer and 1µL unknown DNA sample to tube 7
6. determine absorbance w/ spectrophotometer and plot absorbance vs. concentration data
30
TE Buffer - Tris EDTA
EDTA maintains the integrity of DNA by
inhibiting DNAase via ion chelation
the buffer maintains a consistent pH (7.5) - DNA absorbs differently at 260 nm depending on the pH
31
wavelength at which DNA absorbs light
260 nm
32
restriction enzyme
recognize double-stranded DNA, cleave at specific palindromic recognition sequence - usually 4 or 6 base pairs
33
why don't REs digest their own DNA
their DNA is methylated to prevent digestion
34
EcoRI name
Eco = e. coli
R = R strain of e. coli
I = enzyme number (first isolated strain)
35
EcoRI recognition site
5' GAATTC 3'
cuts between G and A
36
HINDIII recognition site
5' AAGCTT 3'
37
sticky ends
can form hydrogen bonds with complementary ends
38
needed to use restriction enzyme to cut DNA
- template DNA
– Restriction enzyme
– RE buffer specific for that enzyme which allows the enzyme to function
– Heat: The digest will be run at 37 *C
39
PCR (polymerase chain reaction) definition
Site-specific, exponential amplification (2N) of DNA performed in vitro (i.e., in a test tube in the absence of any
living organism)
40
PCR requirements
template DNA
single stranded DNA
deoxynucleotides (dNTPs)
Mg2+
Buffer
DNA polymerase
41
use of single-stranded DNA primers in PCR
primers on each strand of DNA anneal to the template DNA and bookend the piece of DNA that will be amplified
42
use of dNTPs in PCR
building blocks of PCR rxn
43
use of Mg2+ in PCR
binds to negatively charged phosphate groups of DNA and stabilizes rxn
44
use of buffer in PCR
maintains pH required by PCR enzyme (DNA polymerase)
45
use of DNA polymerase in PCR
enzyme which carries out reaction - joins nucleotides together complementary to the template DNA
46
length of PCR primer and why
18-22 bp
longer = binding becomes difficult
shorter = not enough specificity
47
GC content in PCR primer
40-60%
increases stability to increase melting temperature
48
melting temp (half of primers dissociate) determines...
annealing temp (abt 5*C lower)
49
melting temp is determined by
size and GC content of PCR primer
50
3 steps in a PCR rxn
denaturation, annealing, extension
51
denaturation in PCR
usually at 95*C, 30s
allows 2 strands of DNA to dissociate
52
annealing in PCR
usually at 55*C depending on melting temperature - higher % of GC = higher melting temperature
30s
primers bind to template DNA
53
extension in PCR
at 68*C for 50s
DNA polymerase extends daughter strands by adding dNTPs
final extension at 72*C for 10 minutes
54
cause of size separation in DNA agarose gel
larger DNA gets stuck in gel matrix (carb. complex)
small DNA moves more easily
voltage does not cause size separation, just movement
55
agarose gel contains:
sybr green, ions
56
sybr green
intercalator - binds between two DNA strands, is UV reactive and glows to visualize fragments
57
ions in gel
allow current flow through gel
more salt = more current
58
DNA ladder
contains fragment pieces of known size to estimate size of unknown fragments
59
salt-containing buffer in gel
replaces ions that leave the gel due to current
60
purpose of reaction digest
determine the difference in genomic DNA vs smaller DNA (lambda phage) when digested by restriction enzymes
61
small pieces of DNA (ex. lambda phage) appear as _____ on the gel
sharp bands
62
genomic DNA will appear as ____ on the gel
smears
63
linear DNA migrates ______ than circular, because...
slower; circular DNA tends to supercoil, become dense, and move faster
64
probability of getting a 6 base sequence
1/4 ^6
= 1/4096
divide DNA sample size by 4096 to find total number of fragments
65
theoretical yield of DNA after PCR
2^n - n is the number of cycles
66
find total # of DNA molecules
2^n (# molecules to start)
67
issues preventing theoretical yield of DNA after PCR
run out of reagents, thermocycler malfunction, human error
68
needed for PCR amplification of insulin gene
PCR buffer
forward primer
reverse primer
dNTP
taq DNA polymerase
calf genomic DNA
DI water
69
stop PCR amplification of insulin
put on ice (4*C)
70
4 parts of molecular cloning
1. isolate DNA of interest
2. ligate insert into vector
3. transform ligation into bacterial cells
4. screen clones for insert
71
purpose of transforming DNA into bacteria
cells will make copies of plasmid with insulin gene (DNA -> RNA -> protein)
72
ligation reaction steps
annealing and ligation
73
annealing (before ligation)
allowing complementary DNA to form hydrogen bonds (connects complementary sticky ends) to form two strands
74
ligation
formation of a phosphodiester bond between hydroxyl group 3' of one nucleotide and phosphate group 5' of another
75
ligation reagents
T4 DNA ligase
insert (PCR product)
plasmid vector (pGemT)
buffer (contains ATP for energy of DNA ligase)
1 hour at room temperature
76
pGemT vector
plasmid carrying genes that confer resistance to specific antibiotics - pGemT has ampicillin resistance gene
77
pGemT is pre-
linearized
78
features of pGemT
3' T-overhangs in polylinker site (multiple cloning site) containing cutting site for REs
ampicillin resistance gene, LacZ gene (blue/white selection)
79
T-overhang
on pGemT vector; anneals to A overhang that is left by Taq DNA polymerase
80
TA cloning
made possible by annealing A overhang of PCR product and T overhang of vector - most efficient
81
ingredients of bacteria plating for screening
LB (nutrients for bacteria)
Agar
ampicillin
X-Gal/IPTG
82
ampicillin
toxic to bacteria w/o resistance
AmpR (in pGemT) confers resistance
83
LacZ gene
produces protein called beta-galactosidase which can cleave sugars
84
if b-galactosidase cleaves X-gal (lactose-like sugar)...
becomes blue
85
blue colonies
pGemT without insert
86
white colonies
contain pGemT with insert
87
insulin insert will disrupt LacZ gene ->
X-gal will not be cleaved -> white colonies
88
transformation
competent cells (with this ability) take up naked DNA
89
cells we use in transformation
JM109 - altered from E. coli with competent membranes
90
transformation process
1. heat shock cells at 42* C to make them take up DNA - express heat shock proteins that allow them to uptake DNA
2. allow cells to recover in nutrient-rich media (SOC)
91
optimal incubation temp for T4 DNA ligase
4 - 25*C (incubated at room temp for 1 hr)
92
competent cells formed by
treatment w/ calcium chloride in the early log phase of growth
93
IPTG
induces the lac operon (for expression of lacZ gene)
94
non-functional x-gal gene (no XGal sugar)
functional insulin gene disrupted its function in multiple cloning site
95
MCS region within lacZ gene in pGemT ensures...
cloning of a DNA fragment into MCS leads to non-functional X-gal protein
96
OriC
origin where cloning will begin in the vector
97
blue summary
no insert, lacz gene not disrupted, b-galactosidase produced, x-gal broken down
98
white summary
lacz gene disrupted, b-galactosidase not produced, x-gal not cleaved, hopefully contains DNA insert
99
miniprep steps
1. centrifuge colony cells to form pellet, remove supernatant
2. add p1 (resuspension buffer) and mix
3. add p2 (lysis buffer) and mix gently
4. add N3 (neutralization buffer) and mix
5. centrifuge, get pellet, add supernatant to spin column
6. centrifuge column - DNA will stick to column and all else will flowthrough
7. add PB (wash buffer 1) and centrifuge
8. add PE (wash buffer 2) and centrifuge
9. add EB buffer (water)
100
P1 buffer
contains Tris-CL and RNase A
101
Tris-Cl
physiological pH buffer
102
RNase A
enters cell and degrades all RNA - prevents structural similarity of RNA and DNA interfering w/ plasmid isolation later
103
P2 buffer
contains NaOH and SDS
104
NaOH
denatures genomic and plasmid DNA
105
SDS
sodium dodecyl sulfate - amphipathic detergent, denatures the cellular and nuclear membranes to allow DNA to go into the solution
106
N3 buffer
neutralization - contains acidic potassium acetate
high salt conc. precipitates protein and large genomic DNA, not smaller plasmid DNA, which will renature
107
PB buffer
wash buffer 1
removes DNases that could degrade plasmid DNA
contains isopropanol and salts to ensure DNA stays attached to columb
108
PE buffer
wash buffer 2
removes ions and impurities from plasmid DNA
contains ethanol and salts to ensure DNA stays attached to column
109
EB buffer (water)
elution buffer
low salt buffer allows plasmid DNA to exit column - DNA is able to regain negative charge in low-salt environment - no longer attracted to silica column
110
smaller fragments move through gel ____
faster
111
higher concentration of gel will cause _____ migration of fragments
slower
112
higher voltage in gel will make all fragments migrate ______
faster
113
plasmids isolated from BLUE colonies will migrate _______ because they are ________ (they lack _______)
faster; smaller; insert
114
digested white DNA has 2 bands because ....
digestion cut at RE sites (EcoRI)
smaller band is insert; larger band is vector - same size as blue colonies
115
alkaline lysis
DNA is released into solution by disrupting cell membranes - treated w/ alkaline solution to break H-bonds, then neutralized w/ acid - only small plasmid DNA can renature
first few steps in this process
116
silica column affinity
in the presence of cations, plasmid binds to silica column despite both usually having negative charges
allows plasmid to stick to column when washed
117
digested blue
linear DNA w/o insert
3nm
118
undigested blue
3 nm but slightly smaller than digested (lower)
plasmid w/o insert
119
undigested white
linear DNA w/ insert
4nm
highest
120
digested white
2 pieces - vector (3nm) and insert (1nm)
