Lab 23 Goals and Objectives: ***Begin lab before lecture!!! EDVOKIT#124: DNA-based Screening for Smallpox Our DNA samples: -collect patient sample (blood, swab, etc.) -purify DNA -set up PCR using primers specific to suspect pathogen -our primers = match a sequence for pox virus genome DNA, -monkey pox and small pox have different genes in between, thus different sized genomes: Monkey Pox = 1038bp between primers Small Pox = 1948bp between primers -run PCR results on gel, use size to determine which virus our patient has

DNA Gel Electrophoresis -place in running chamber with electrolyte buffer -electrical current runs through buffer between electrodes on opposite sides of gel + -

-DNA samples loaded into wells near negative electrode - DNA must be suspended in loading buffer: 1. glycerol to make sample dense to sink through running buffer into well 2. two tracking dyes to follow movement through gel (DNA is colorless) -bromophenol blue: co-migrates with ~300bp (small DNA) -xylene cyanol: co-migrates with ~4000bp (big DNA)

-DNA has negative charge due to to phosphate backbone -DNA moves through gel away from negative toward positive electrode -gel matrix separates moving DNA by size: -smaller molecules “squeeze” through gel easier thus moving faster -smaller molecules end up further away from the wells -DNA will need to be stained to see it after running the gel + - Bigger Smaller

PCR must have controls! 1. Molecular weight marker 2. purified small pox DNA: confirms primers work on small pox to produce expected size, shows expected product(s) (positive control) 3. Patient sample 4. purified monkey pox DNA: confirms primers work on monkey pox to produce expected size, shows expected product(s) (positive control) 5. uninfected human DNA: confirms primers do not produce products with human DNA (or if they do, we know what to ignore) (negative control) MW SP Patient MP C 

***Begin lab before lecture!!! 1. Add loading buffer (dye) to PCR samples and load as much of each sample as will fit in the well into the good experimental gel: !!!! no air bubbles in tip while loading! 2. Be certain power source is set to 70 VOLTS not AMPS Run gels for ~1.5 hrs (have lecture while waiting) 4. After gel runs, DNA must be stained with methylene blue to visualize it - Stain and destain gel 5. Interpret results