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PCR Activity
Zippers are used to visually
demonstrate how PCR can take
one segment of DNA and create 8
copies
Ingredients needed for PCR
 Master-mix + DNA
 PNM (primer nucleotide mix)
 10x incubation buffer, MgCl2, Sterile
molecular grade water
 DNA polymerase (HotStarTaq)
 DNA sample
Where does PCR happen?
 Inside small 0.2 mL
reaction tubes
 A thermocycler is a
programmable
instrument that will
heat and cool the
reaction tubes to the
desired temperature
for a designated
amount of time.
Cycle 1 -Step 1. Denaturation
 Temperature:
95º C for 25
seconds
 At 95º C the double
strand of DNA
separates
 Unzip the DNA
Cycle 1 -Step 2: Annealing
 Temperature:
53º C for 40
seconds
 Primers hybridize to
complementary
sequences of each
DNA strand.
 Place the primer
label onto the edge
of the zipper
Cycle 1 -Step 3: Extension
 Temperature:
70º C for 40
seconds
 Taq polymerase adds
nucleotides to the 3’ end
of each primer
 Locate the
complimentary
nucleotide “label” and
stick it on the edge of
the zipper opposite the
original nucleotide and
zip it together as each
nucleotide is added
 Complements = A-T and
G-C
After 1 cycle
Cycle 2 -Step 1. Denaturation
 Temperature:
95º C for 25
seconds
 At 95º C the double
strand of DNA
separates
 Unzip the DNA
 Pass a copy to your
neighbor
Cycle 2 -Step 2: Annealing
 Temperature:
53º C for 40
seconds
 Primers hybridize to
complementary
sequences of each
DNA strand.
 Place the primer
label onto the edge
of the zipper
Cycle 3 -Step 3: Extension
 Temperature:
70º C for 40
seconds
 Taq polymerase adds
nucleotides to the 3’ end
of each primer
 Locate the
complimentary
nucleotide “label” and
stick it on the edge of
the zipper opposite the
original nucleotide and
zip it together as each
nucleotide is added
After 2 cycles
Cycle 3 -Step 1. Denaturation
 Temperature:
95º C for 25
seconds
 At 95º C the double
strand of DNA
separates
 Unzip the DNA
 Pass a copy to your
neighbor
Cycle 3 -Step 2: Annealing
 Temperature:
53º C for 40
seconds
 Primers hybridize to
complementary
sequences of each
DNA strand.
 Place the primer
label onto the edge
of the zipper
Cycle 3 -Step 3: Extension
 Temperature:
70º C for 40
seconds
 Taq polymerase adds
nucleotides to the 3’ end
of each primer
 Locate the
complimentary
nucleotide “label” and
stick it on the edge of
the zipper opposite the
original nucleotide and
zip it together as each
nucleotide is added
After 3 cycles
End Result: Exponential amplification
of a target sequence of DNA

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IV PCR Activity 010510.ppt

  • 1. PCR Activity Zippers are used to visually demonstrate how PCR can take one segment of DNA and create 8 copies
  • 2. Ingredients needed for PCR  Master-mix + DNA  PNM (primer nucleotide mix)  10x incubation buffer, MgCl2, Sterile molecular grade water  DNA polymerase (HotStarTaq)  DNA sample
  • 3. Where does PCR happen?  Inside small 0.2 mL reaction tubes  A thermocycler is a programmable instrument that will heat and cool the reaction tubes to the desired temperature for a designated amount of time.
  • 4. Cycle 1 -Step 1. Denaturation  Temperature: 95º C for 25 seconds  At 95º C the double strand of DNA separates  Unzip the DNA
  • 5. Cycle 1 -Step 2: Annealing  Temperature: 53º C for 40 seconds  Primers hybridize to complementary sequences of each DNA strand.  Place the primer label onto the edge of the zipper
  • 6. Cycle 1 -Step 3: Extension  Temperature: 70º C for 40 seconds  Taq polymerase adds nucleotides to the 3’ end of each primer  Locate the complimentary nucleotide “label” and stick it on the edge of the zipper opposite the original nucleotide and zip it together as each nucleotide is added  Complements = A-T and G-C
  • 8. Cycle 2 -Step 1. Denaturation  Temperature: 95º C for 25 seconds  At 95º C the double strand of DNA separates  Unzip the DNA  Pass a copy to your neighbor
  • 9. Cycle 2 -Step 2: Annealing  Temperature: 53º C for 40 seconds  Primers hybridize to complementary sequences of each DNA strand.  Place the primer label onto the edge of the zipper
  • 10. Cycle 3 -Step 3: Extension  Temperature: 70º C for 40 seconds  Taq polymerase adds nucleotides to the 3’ end of each primer  Locate the complimentary nucleotide “label” and stick it on the edge of the zipper opposite the original nucleotide and zip it together as each nucleotide is added
  • 12. Cycle 3 -Step 1. Denaturation  Temperature: 95º C for 25 seconds  At 95º C the double strand of DNA separates  Unzip the DNA  Pass a copy to your neighbor
  • 13. Cycle 3 -Step 2: Annealing  Temperature: 53º C for 40 seconds  Primers hybridize to complementary sequences of each DNA strand.  Place the primer label onto the edge of the zipper
  • 14. Cycle 3 -Step 3: Extension  Temperature: 70º C for 40 seconds  Taq polymerase adds nucleotides to the 3’ end of each primer  Locate the complimentary nucleotide “label” and stick it on the edge of the zipper opposite the original nucleotide and zip it together as each nucleotide is added
  • 16. End Result: Exponential amplification of a target sequence of DNA