pcr

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

7. After 1 cycle

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

11. After 2 cycles

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

15. After 3 cycles

16. End Result: Exponential amplification
of a target sequence of DNA