Download Lab23

Lab 23 Goals and Objectives:
***Begin lab before lecture!!!
EDVOKIT#124: DNA-based Screening for Smallpox
Practice loading samples into “submarine gels” with practice
gel
Add 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 AMPS
Run gels for ~1.5 hrs (have lecture while waiting)
Stain and destain gel
Interpret 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 primers
Small 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 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)
-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 Smallpox
Practice loading samples into “submarine gels” with practice
gel
Add 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 AMPS
Run gels for ~1.5 hrs (have lecture while waiting)
Stain and destain gel
Interpret results