PCR

1
PCR

• Bibi Haniff April Williams Laurie Brier

2
PCRWhats Required

• Template DNA
• Forward Primer
• Reverse Primer
• dNTPs
• Taq DNA Polymerase
• Reaction BufferMgCl2
• Water
• PCR Machine

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PCRWhats Optional

• Pfu Polymerase
• Bovine Serum Albumin
• Spermidine
• Glycerol

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Whats a Primer?

• Promotes initiation of replication
• Free 3 OH
• Complementary to template
• DNA elongation

5
Before you start PCR

• Collect Cells
• Purify Cells for PCR
• Instagene Matrix
• Lyse Cells
• Release of DNA from Cell
• Heat

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PCR in 3 Steps

• Denaturation
• Reaction mixture is heated to 94C
• Annealing
• Oligonucleotide primers anneal to complimentary
  sequences on templates
• at 60C
• Extenstion
• Taq DNA polymerase adds nucleotides A,T,C,and G
  to create complimentary
• copy at 72C

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Summary
8
Alu Sequences

• Repetitive DNA elements found throughout the
  human genome
• Consisting of 300 Base Pairs
• Chromosome 16
• What is the function of Alu?

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PCR Results

• The amount of DNA that you have doubles per
  cycle.

http//www.aaranyak.org/Projects/PCR.htm
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Problem 1

• If you start off with 1 strand of DNA and run the
  PCR for 5 cycles how many strands of DNA do you
  end up with?
• Answer 32 strands of DNA

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Problem 2

• If you start with 5 strands of DNA and run the
  PCR for a standard 30 cycles.how much DNA is
  produced?

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Math behind Amplification

• N 2t(N0)
• Where
• N of copies after amplification
• t the of cycles
• N0 the of targeted DNA sequences in the
  sample prior to running PCR

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Applying the Math

• N 2t(N0)
• N 230(5)
• ANSWER 5,368,709,120 strands

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Works Cited

• Roche Applied Science. PCR Applications Manual.
  3rd Edition.
• Seidman LA.2008. Basic laboratory calculations
  for biotechnology. San Fransisco (CA) Pearson
  Benjamin Cummings. 472 p