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Lab 22 Goals and Objectives:
EDVOKIT#300: Blue/White Cloning of a DNA Fragment
Calculate transformation efficiencies
*Control = transformation efficiency of cells to take up intact
plasmid
*All ligation colonies = ligation efficiency
*White ligation colonies = cloning efficiency
Exercise 65 and Handout: PCR
Five (5) groups possible!
Same group for PCR and EDVOKIT #124
Set up PCR reactions (supplement pg 97-98) and place in
thermocycler
EDVOKIT#124: DNA-based Screening for Smallpox
Prepare DNA agarose gels, 2 per group (Edvo pgs 8-9):
1 practice gel with three combs
1 experimental gel with one comb
Wrap gels and store until next lab
***Next lab gels will need to run for 1.5hrs so we will load our PCR
samples and begin running the gel BEFORE lecture!
count
colonies to
calculate
efficiency:
colonies
per µg of
DNA
all colonies =
ligation efficiency
transformation
efficiency
white colonies =
cloning efficiency
Control
Ligation
Control
Ligation
Control
Ligation
Control
Ligation
Polymerase Chain Reaction (PCR)
Method for amplifying/copying DNA
-can turn one copy into 1 X 109 copies in ~3hr
-generate large quantities for study:
1. determine crime suspect from minute traces of
body fluids/tissues
2. detect infectious disease
3. gene cloning (to extract gene from source)
Minimum requirements:
1. Template DNA (source you want to copy)
2. DNA polymerase (enzyme to synthesize new DNA)
3. Primers (ssDNA fragments, complementary to ends
of target DNA sequence)
4. Free nucleotides (ATP, CTP, TTP, GTP)
DNA Synthesis in a tube (PCR)
1. Double stranded DNA template must
be separated
2. DNA primers base pair to ends of
single stranded target sequence
3. DNA polymerase adds nucleotides to
the 3’ end of the primers by complementary
base pairing free nucleotides to the template strand
4. Repeat steps 1-3: each new copy generated can
serve as a template in the next round of replication
*1 DNA molecule X 30 rounds of replication =
1.1 X 109 molecules
(1073741824)
“Round of Replication” or cycle conditions (supp. pg 98)
-in living cells (in vivo), enzymes and stabilizing proteins
assist the DNA polymerase in the synthesis process
-in a tube (in vitro), temperature is varied to facilitate the
necessary molecular events in the absence of life
Hot Start - “melt” all the molecules and reagents (unstick,
unclump) and mix them together, done once at the beginning
Cycle:
95°C, 30sec = denaturation: separate the double stranded
DNA template (break H-bonds)
50-60°C, 30sec = annealing: allows primers to complementary
base pair with target sequence on template DNA, temp
varied depending on %GC content
72°C, 1min/kb DNA = extension: optimal temp for DNA
polymerase to synthesize new DNA molecules from ends of
primers
Repeat cycle 20-30 times; products from one round serve as
templates in the next
95°C, 30sec = denaturation:
separate the double stranded
DNA template (break H-bonds)
50-60°C, 30sec = annealing:
allows primers to
complementary base pair with
target sequence on template
DNA
72°C, 1min/kb DNA = extension:
optimal temp for DNA
polymerase to synthesize new
DNA molecules from ends of
primers
Denature
Annealing
Extension
Figure 65.3
Perform in a thermocycler
Use heat stable DNA polymerase: Taq
Lab 22 Goals and Objectives:
EDVOKIT#300: Blue/White Cloning of a DNA Fragment
Calculate transformation efficiencies
*Control = transformation efficiency of cells to take up intact
plasmid
*All ligation colonies = ligation efficiency
*White ligation colonies = cloning efficiency
Exercise 65 and Handout: PCR
Five (5) groups possible!
Same group for PCR and EDVOKIT #124
Set up PCR reactions (supplement pg 97-98) and place in
thermocycler
EDVOKIT#124: DNA-based Screening for Smallpox
Prepare DNA agarose gels, 2 per group (Edvo pgs 7-9):
1 practice gel with three combs
1 experimental gel with one comb
Wrap gels and store until next lab
***Next lab gels will need to run for 1.5hrs so we will load our PCR
samples and begin running the gel BEFORE lecture!