Lab Exam 1 Flashcards
week 1 lab goal
extract DNA from calf thymus tissue
week 2 lab goal
quantify extracted DNA (concentration)
week 3 lab goal
PCR to amplify insulin gene
week 4 lab goal
ligate insulin gene into plasmid, transform into bacterial cells
week 5 lab goal
analyze transformants w/ gel
3 reasons calf thymus is beneficial
- 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
molecular cloning
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
steps of DNA extraction w/ silica membrane
(lab 1) solution for DNase, enzyme that destroys DNA
use EDTA - chelating agent that binds
with cations such as Ca and Mg needed by DNase and forms a water-soluble compound
(lab 1) solution to stop other proteins mixing with DNA
Proteinase K digests proteins, sodium
dodecyl sulfate (SDS) is a surfactant that lyses cells
(lab 1) solution extreme pH environment (DNA is most stable in a neutral pH range)
Tris-HCl or PBS - buffer solutions used throughout the experiment to avoid
drastic pH fluctuations
proteinase K
digests proteins
Buffer ATL
contains SDS and the chelating agent EDTA
SDS
detergent used to lyse cells by disrupting non-covalent bonds in protein and denatures them
EDTA
chelating agent - bonds with Mg and Ca to prevent DNase from getting them
Buffer AL
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
Buffer AW1 and AW2
wash buffers that keep the DNA bound to the spin column while washing DNA of contaminants and impurities
DNA elution buffer
water - low in salt, releases DNA from spin column
procedure of lab 1
- 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
optimal temp for proteinase K function
56* C
1000 microliters
1 mL
AW1 components
guanidine hydrochloride
ethanol
AW2 components
ethanol
guanidine hydrochloride
chaotropic salt - high salt content increases DNA binding to silica membrane, + denatures proteins
Beer’s Law
there is a direct linear relationship between the optical absorbance (A) of a
compound and its concentration
A=kc
A = absorbance
k = slope of line of concentration/absorbance graph
c = concentration
spectrophotometer
determines absorption of light at a specific wavelength/range of wavelengths
k value
50 on standard lab spectrophotometers measuring DNA concentration
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
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
wavelength at which DNA absorbs light
260 nm
restriction enzyme
recognize double-stranded DNA, cleave at specific palindromic recognition sequence - usually 4 or 6 base pairs
why don’t REs digest their own DNA
their DNA is methylated to prevent digestion
EcoRI name
Eco = e. coli
R = R strain of e. coli
I = enzyme number (first isolated strain)
EcoRI recognition site
5’ GAATTC 3’
cuts between G and A
HINDIII recognition site
5’ AAGCTT 3’
sticky ends
can form hydrogen bonds with complementary ends
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
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)
PCR requirements
template DNA
single stranded DNA
deoxynucleotides (dNTPs)
Mg2+
Buffer
DNA polymerase
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
use of dNTPs in PCR
building blocks of PCR rxn
use of Mg2+ in PCR
binds to negatively charged phosphate groups of DNA and stabilizes rxn
use of buffer in PCR
maintains pH required by PCR enzyme (DNA polymerase)
use of DNA polymerase in PCR
enzyme which carries out reaction - joins nucleotides together complementary to the template DNA
length of PCR primer and why
18-22 bp
longer = binding becomes difficult
shorter = not enough specificity
GC content in PCR primer
40-60%
increases stability to increase melting temperature
melting temp (half of primers dissociate) determines…
annealing temp (abt 5*C lower)
melting temp is determined by
size and GC content of PCR primer
3 steps in a PCR rxn
denaturation, annealing, extension
denaturation in PCR
usually at 95*C, 30s
allows 2 strands of DNA to dissociate
annealing in PCR
usually at 55*C depending on melting temperature - higher % of GC = higher melting temperature
30s
primers bind to template DNA
extension in PCR
at 68C for 50s
DNA polymerase extends daughter strands by adding dNTPs
final extension at 72C for 10 minutes
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
agarose gel contains:
sybr green, ions
sybr green
intercalator - binds between two DNA strands, is UV reactive and glows to visualize fragments
ions in gel
allow current flow through gel
more salt = more current
DNA ladder
contains fragment pieces of known size to estimate size of unknown fragments
salt-containing buffer in gel
replaces ions that leave the gel due to current
purpose of reaction digest
determine the difference in genomic DNA vs smaller DNA (lambda phage) when digested by restriction enzymes
small pieces of DNA (ex. lambda phage) appear as _____ on the gel
sharp bands
genomic DNA will appear as ____ on the gel
smears
linear DNA migrates ______ than circular, because…
slower; circular DNA tends to supercoil, become dense, and move faster
probability of getting a 6 base sequence
1/4 ^6
= 1/4096
divide DNA sample size by 4096 to find total number of fragments
theoretical yield of DNA after PCR
2^n - n is the number of cycles
find total # of DNA molecules
2^n (# molecules to start)
issues preventing theoretical yield of DNA after PCR
run out of reagents, thermocycler malfunction, human error
needed for PCR amplification of insulin gene
PCR buffer
forward primer
reverse primer
dNTP
taq DNA polymerase
calf genomic DNA
DI water
stop PCR amplification of insulin
put on ice (4*C)
4 parts of molecular cloning
- isolate DNA of interest
- ligate insert into vector
- transform ligation into bacterial cells
- screen clones for insert
purpose of transforming DNA into bacteria
cells will make copies of plasmid with insulin gene (DNA -> RNA -> protein)
ligation reaction steps
annealing and ligation
annealing (before ligation)
allowing complementary DNA to form hydrogen bonds (connects complementary sticky ends) to form two strands
ligation
formation of a phosphodiester bond between hydroxyl group 3’ of one nucleotide and phosphate group 5’ of another
ligation reagents
T4 DNA ligase
insert (PCR product)
plasmid vector (pGemT)
buffer (contains ATP for energy of DNA ligase)
1 hour at room temperature
pGemT vector
plasmid carrying genes that confer resistance to specific antibiotics - pGemT has ampicillin resistance gene
pGemT is pre-
linearized
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)
T-overhang
on pGemT vector; anneals to A overhang that is left by Taq DNA polymerase
TA cloning
made possible by annealing A overhang of PCR product and T overhang of vector - most efficient
ingredients of bacteria plating for screening
LB (nutrients for bacteria)
Agar
ampicillin
X-Gal/IPTG
ampicillin
toxic to bacteria w/o resistance
AmpR (in pGemT) confers resistance
LacZ gene
produces protein called beta-galactosidase which can cleave sugars
if b-galactosidase cleaves X-gal (lactose-like sugar)…
becomes blue
blue colonies
pGemT without insert
white colonies
contain pGemT with insert
insulin insert will disrupt LacZ gene ->
X-gal will not be cleaved -> white colonies
transformation
competent cells (with this ability) take up naked DNA
cells we use in transformation
JM109 - altered from E. coli with competent membranes
transformation process
- heat shock cells at 42* C to make them take up DNA - express heat shock proteins that allow them to uptake DNA
- allow cells to recover in nutrient-rich media (SOC)
optimal incubation temp for T4 DNA ligase
4 - 25*C (incubated at room temp for 1 hr)
competent cells formed by
treatment w/ calcium chloride in the early log phase of growth
IPTG
induces the lac operon (for expression of lacZ gene)
non-functional x-gal gene (no XGal sugar)
functional insulin gene disrupted its function in multiple cloning site
MCS region within lacZ gene in pGemT ensures…
cloning of a DNA fragment into MCS leads to non-functional X-gal protein
OriC
origin where cloning will begin in the vector
blue summary
no insert, lacz gene not disrupted, b-galactosidase produced, x-gal broken down
white summary
lacz gene disrupted, b-galactosidase not produced, x-gal not cleaved, hopefully contains DNA insert
miniprep steps
- centrifuge colony cells to form pellet, remove supernatant
- add p1 (resuspension buffer) and mix
- add p2 (lysis buffer) and mix gently
- add N3 (neutralization buffer) and mix
- centrifuge, get pellet, add supernatant to spin column
- centrifuge column - DNA will stick to column and all else will flowthrough
- add PB (wash buffer 1) and centrifuge
- add PE (wash buffer 2) and centrifuge
- add EB buffer (water)
P1 buffer
contains Tris-CL and RNase A
Tris-Cl
physiological pH buffer
RNase A
enters cell and degrades all RNA - prevents structural similarity of RNA and DNA interfering w/ plasmid isolation later
P2 buffer
contains NaOH and SDS
NaOH
denatures genomic and plasmid DNA
SDS
sodium dodecyl sulfate - amphipathic detergent, denatures the cellular and nuclear membranes to allow DNA to go into the solution
N3 buffer
neutralization - contains acidic potassium acetate
high salt conc. precipitates protein and large genomic DNA, not smaller plasmid DNA, which will renature
PB buffer
wash buffer 1
removes DNases that could degrade plasmid DNA
contains isopropanol and salts to ensure DNA stays attached to columb
PE buffer
wash buffer 2
removes ions and impurities from plasmid DNA
contains ethanol and salts to ensure DNA stays attached to column
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
smaller fragments move through gel ____
faster
higher concentration of gel will cause _____ migration of fragments
slower
higher voltage in gel will make all fragments migrate ______
faster
plasmids isolated from BLUE colonies will migrate _______ because they are ________ (they lack _______)
faster; smaller; insert
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
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
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
digested blue
linear DNA w/o insert
3nm
undigested blue
3 nm but slightly smaller than digested (lower)
plasmid w/o insert
undigested white
linear DNA w/ insert
4nm
highest
digested white
2 pieces - vector (3nm) and insert (1nm)
