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EDVOKIT#300: Blue/White Cloning of a DNA Fragment
Lab 22 Goals and Objectives: EDVOKIT#300: Blue/White Cloning of a DNA Fragment Calculate transformation efficiencies Compare 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 #124 Set up PCR reactions (supplement pg 89-90) and place in thermocycler EDVOKIT#124: DNA-based Screening for Smallpox Prepare DNA agarose gels, 2 per group (pg 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!
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count colonies to calculate efficiency: colonies per µg of DNA
all colonies = ligation efficiency white colonies = cloning efficiency transformation efficiency
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Control Ligation Control Ligation Control Ligation Control Ligation
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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)
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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( ) Fig.67.1
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“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 life Hot Start - “melt” all the molecules and reagents (unstick, unclump) and mix them together, done once at the beginning 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 times
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Fig.67.1 Perform in a thermocycler Use heat stable DNA polymerase: Taq
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EDVOKIT#300: Blue/White Cloning of a DNA Fragment
Lab 22 Goals and Objectives: EDVOKIT#300: Blue/White Cloning of a DNA Fragment Calculate transformation efficiencies Compare 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 #124 Set up PCR reactions (supplement pg 89-90) and place in thermocycler EDVOKIT#124: DNA-based Screening for Smallpox Prepare DNA agarose gels, 2 per group (pg 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!
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