Lab 22 Goals and Objectives:
EDVOKIT#300: Blue/White Cloning of a DNA FragmentCalculate transformation efficienciesCompare control efficiency to cloned gene (white colony) efficiency
*Control = transformation efficiency of cells to take up intact plasmid*All ligation colonies = transformation efficiency + ligation efficiency*White ligation colonies = transformation efficiency plus efficiency of
ligation of desired gene into plasmid (cloning efficiency)Exercise 67 and Handout: PCR
**Five (5) groups possible! Same group for PCR and EDVOKIT #124Set up PCR reactions (supplement pg 89-90) and place in thermocycler
EDVOKIT#124: DNA-based Screening for SmallpoxPrepare DNA agarose gels, 2 per group (pg 7-9):
1 practice gel with three combs1 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
transformation efficiency
all colonies = ligation efficiency
white colonies = cloning efficiency
Control Ligation Control Ligation
Control LigationLigationControl
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 disease3. 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 separated2. DNA primers base pair to ends of
single stranded target sequence3. 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)
Fig.67.1
“Round of Replication” or cycle conditions (supp. pg.90)-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 lifeHot Start - “melt” all the molecules and reagents (unstick,
unclump) and mix them together, done once at the beginning95°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
Fig.67.1
Perform in a thermocycler
Use heat stable DNA polymerase: Taq
Lab 22 Goals and Objectives:
EDVOKIT#300: Blue/White Cloning of a DNA FragmentCalculate transformation efficienciesCompare control efficiency to cloned gene (white colony) efficiency
*Control = transformation efficiency of cells to take up intact plasmid*All ligation colonies = transformation efficiency + ligation efficiency*White ligation colonies = transformation efficiency plus efficiency of
ligation of desired gene into plasmid (cloning efficiency)Exercise 67 and Handout: PCR
**Five (5) groups possible! Same group for PCR and EDVOKIT #124Set up PCR reactions (supplement pg 89-90) and place in thermocycler
EDVOKIT#124: DNA-based Screening for SmallpoxPrepare DNA agarose gels, 2 per group (pg 7-9):
1 practice gel with three combs1 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!
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