1. 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 = 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 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!

2. Control Ligation EcoR1 DNA fragmentEcoR1 Digested with EcoR1 Ligase Transformation Two possible products: -gene ligated into vector -vector religated without gene transformation efficiency all colonies = ligation efficiency white colonies = cloning efficiency Count colonies to calculate cloning efficiency (colonies per µg of DNA used in transfection experiment) – The quantity of DNA used in Ligation reaction was 0.012 μg Number of transformants μg of DNA used in transformation

3. Control Ligation Control Ligation Control Ligation Control

4. Polymerase Chain Reaction (PCR) Method for amplifying/copying DNA -can turn one copy into 1 X 10 9 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 for polymerase reaction: 1. Template DNA (the target molecule to be copy) 2. Primers (ssDNA molecules, complementary to specific sequence of thetarget DNA) 3. Free nucleotides (ATP, CTP, TTP, GTP needed for synthesis of the new DNA chain) 4. Buffer (supply with optimal condition for Taq Polymerase) 5. Taq polymerase (enzyme to synthesize new DNA molecules)

5. Use heat stable DNA polymerase:Taq polymerase Perform in a thermocycler 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

6. PCR Figure 9.4.1 * 1 DNA molecule X 30 rounds of replication = 1.1 X 109 molecules(1073741824)

7. Hot Start - 95ºC for 5min “melt” all the molecules and reagents (unstick, unclump) and mix them together, done once at the beginning 1. Denaturation - 95ºC, 30sec : separate the double stranded DNA template (break H-bonds) 2. Annealing - 50-60ºC, 30sec : allows primers to complementary base pair with target sequence on template DNA, temperature varied depending on %GC content 3. Extension - 72ºC, 1min/kb DNA : optimal temp for DNA polymerase to synthesize new DNA molecules from ends of primers Repeat cycle 20-30 times

8. Primer1 Primer 2 1.Denaturation 2.Annealing 3.Extention

9. 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!