EDVOKIT#300: Blue/White Cloning of a DNA Fragment

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This presentation was originally prepared by C. William Birky, Jr. Department of Ecology and Evolutionary Biology The University of Arizona It may be used.

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Presentation transcript:

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 *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 white colonies = cloning efficiency transformation 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

Denature Annealing Extension Figure 65.3 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

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 *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!