Download Alu electrophoresis PCR lab

Collect Buccal Cells
PCR
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Polymerase Chain Reaction
DNA/gene amplification
Chromosome 8 and Alu element
Alu + form
Alu - form
Preparing an Agarose Gel for
Electrophoresis
Reagents and Supplies for Gel
Electrophoresis
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Scale - for weighing agarose
Spatula – for scooping agarose powder
Flask or beaker - for melting agarose
Graduated cylinder for measuring buffer
Microwave or hot plate and large beaker to boil water
Agarose
Buffer
Gel tray(s) and comb(s)
Gel box(es)
Power supply
DNA Staining solution
Photo doc. system
Reagents and Supplies for Gel
Electrophoresis Continued
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Your DNA Sample
Loading dye- for help with loading the gel
DNA Ladder or Marker
20uL Pipettes
Tips
Gloves
Waste container
Terms
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Agarose is a polysaccharide (carbohydrate)
polymer material, generally extracted from seaweed.
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Gel electrophoresis is a method that uses an
electrical current and a gel matrix to separate
molecules like DNA and proteins.
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Buffer a solution containing either a weak acid and
its salt or a weak base and its salt, which is resistant
to changes in pH.
Terms continued
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Ethidium Bromide a fluorescent chemical that intercalates
between base pairs in a double stranded DNA molecule.
Commonly used to detect DNA following gel electrophoresis.
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DNA Ladder consists of known DNA sizes used to determine
the size of an unknown DNA sample. The DNA ladder usually
contains regularly spaced sized samples which when run on an
agarose gel looks like a "ladder".
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Power Supply a source of electric current
Percent (%) Solutions
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Percent Weight/Volume (w/v) solutions are prepared
by dissolving a solute usually a solid material into
100 mL of a solution such as water, buffer, DMSO,
etc. Some solutes are not easily dissolved in
solvents so you may need to stir, heat, sonicate or
vortex the solution until it dissolves.
Other types of % solutions are Weight/Weight (W/W)
and Volume/Volume (V/V) solutions
Preparing a 2% W/V Agarose Gel
2% W/V agarose = 2 grams agarose dissolved in
100 milliliters (mL) of Buffer
 For our purposes each gel tray requires 35 mLs of
gel
 To prepare 35 ml of a 2% agarose gel
2 grams
= X grams
100mls
35 mls
X = 0.75 grams agarose
 Weight 0.75 grams agarose and dissolve in 35 mls
buffer
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Directions for preparing and loading
Gel
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Weigh out amount of agarose, measure volume of
buffer needed and place in flask or beaker
Microwave or use hot plate to dissolve agarose
note: do not let melted agarose sit for too long, it will solidify in the
beaker!
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Place comb into casting tray, raise dams on both
ends of tray and pour gel into casting tray
Allow gel to solidify
While gel is solidifying add 4ul of loading dye to your
DNA in your PCR tube
Pour buffer into gel box (~250 mL)
Directions for preparing, loading and
running Gel continued
After gel solidifies:
 Carefully remove comb from gel, put dams down on both ends
of the gel tray
 Place gel tray into gel box with buffer
 Load 10uL of the Marker usually in lane 1 of each gel and your
DNA sample 20uL per well. Once everyone has loaded their
DNA sample plug red electrode to red and black electrode to
black on power supply, make sure the power is turned off on
power supply before connecting electrodes!
 Adjust voltage to 125-135 volts allow gel to run at least 15
minutes, 30 minutes is ideal
Directions continued
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After gel has run for at least 15 minutes, turn off
power supply
Unplug electrodes
Place gel into tray and pour gel staining solution over
gel. The entire gel should be covered with solution
Place tray on rocker for 15 to 30 minutes
Take gel in tray to photodoc station
Place gel on trans illuminator and take picture
Record your genotype in your lab notebook
Gel Electrophoresis
Gel Electrophoresis
Chromosome and Alu element
Alu + form
Alu - form
Alu Allele
on Chomosome 8
#
Genotypes
Alu+
Alu-
Total Alu+
Alleles:
Total AluAlleles:
Alu+/Alu+
Alu+/AluAlu-/AluClass
Total:
Total of both alleles: