PCR Lab: Polymerase Chain Reaction Lab
Objective: To collect a DNA sample from the cells inside a person’s mouth and use this
sample to explore Polymerase Chain Reaction (PCR).
Harvest cheek cells
Transfer the cells to a
microfuge tube
The solution in the
microfuge contains
Chelex Mg++
Helps reduce
the degradation
of DNA
Source of DNA
Centrifuge
BOIL 5
minutes
Separates DNA and cell
organelles by density.
Boiling lyses
the cell
membrane
and releases
DNA.
DNA is in the supernate
liquid on top.
Grab DNA sample
Remove the DNA from the liquid at the top of
the microfuge tube. Transfer to PCR tube.
PCR tube is ready for the Thermocycler.
PCR Thermocycler: Molecules in each PCR tube:
 Your DNA strands (to be copied)
 Free DNA nucleotides
 Taq Polymerase (bacterial heat stable DNA polymerase)
 Primers (frames the locus/site to be copied)
 Buffer (stabilizes pH at 7.4 which is the optimum for enzymes in this lab)
PCR Steps in Thermocycler
(repeated for 24 hours):
1. Heating:
Denatures and Separates DNA strands so the Taq Polymerase can
attach and make copies
2. Annealing:
Primers anneal – attach to DNA to show Taq Polymerase the
starting points.
3. Elongation:
Taq Polymerase attaches at Primers and moves along the DNA
strands to make more copies of the original genes by attaching free
DNA nucleotides according to the Base Pair Rule: A - T
G - C.
After the PCR thermocycler has amplified (made copies) your DNA, students
load DNA sample into the gel wells.
Well #1 will be used for the marker. Students will use the remaining wells.
Record student ID for each well number.
Gel with DNA
Samples
Gel after run the test
IDEAL Gel: MARKER base pairs on left in lane # 1. Three Subjects Data in red:
__
1000
500
400
bp
300
200
100
Left-over primers
Ignore this line
+
Alu- / AluHomo -
Alu+ / Alu+
Homo+
Alu- / Alu+
Heterozygous
Class Sample Data Analysis:
Well Lanes three subjects data:
#3 Heterozygous Alu+Alu#4 Homozygous - Alu#6 Homozygous + Alu+
#6 Heterozygous –
400 bp
100 bp
p
Genotypes
Alu+ / Alu+
Homozygous +
1 in sample
Alu+ / AluHeterozygous
1 in sample
Alu- / AluHomozygous –
1 in sample
Alu+ alleles*
2
0
1
1
0
2
3
Total Alu+ alleles
# of each allele
# of total alleles
Alu- alleles
3
Total Alu- alleles
X 100 = % Frequency
 The term “alleles” refers to forms of a gene or DNA sequence.
 Alu- (100 bp) or Alu+ (400 bp) are the possible alleles here.