Lab 23 Goals and Objectives:

1
Lab 23 Goals and Objectives Begin lab
before lecture!!! EDVOKIT124 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
2
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
3
Bigger

Smaller
4
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
5
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)
6
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
7
Lab 23 Goals and Objectives Begin lab
before lecture!!! EDVOKIT124 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