Lab 23 Goals and Objectives:

***Begin lab before lecture!!!

EDVOKIT#124: DNA-based Screening for SmallpoxPractice loading samples into “submarine gels” with practice

gelAdd 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!

Be certain power source is set to 70 VOLTS not AMPSRun gels for ~1.5 hrs (have lecture while waiting)Stain and destain gelInterpret results

DNA Gel Electrophoresis-use to separate DNA by size to visualize it-Agarose gel = matrix with pores-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 has negative charge due 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

Our DNA samples:-collect patient sample (blood, swab, etc.)-purify DNA-set up PCR using primers specific to suspect pathogen-our primers = pox virus primers,

match ends of pox virus genome-monkey pox and small pox have different genes in

between, thus different sized genomes:Monkey Pox = 1038bp between primersSmall Pox = 1948bp between primers

-run PCR results on gel, use size to determine which virus patient has

PCR must have controls!1. purified small pox DNA: confirms primers

work on small pox to produce expected size, shows expected product(s) (positive control)

2. purified monkey pox DNA: confirms primers work on monkey pox to produce expected size, shows expected product(s) (positive control)

3. uninfected human DNA: confirms primers do not produce products with human DNA (or if they do, we know what to ignore) (negative control)

Agarose gel electrophoresis = “submarine gel”-submerged in running buffer-DNA must be suspended in loading buffer:

contains: 1. glycerol to make sample dense to

sink through running buffer into well2. 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)-After gel runs, DNA must be stained with

methylene blue to visualize it

Lab 23 Goals and Objectives:

***Begin lab before lecture!!!

EDVOKIT#124: DNA-based Screening for SmallpoxPractice loading samples into “submarine gels” with practice

gelAdd 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!

Be certain power source is set to 70 VOLTS not AMPSRun gels for ~1.5 hrs (have lecture while waiting)Stain and destain gelInterpret results