Objectives:To amplify a 500bp fragment of lambda DNA. To understand the principles and the application of Polymerase Chain Reaction. To analyses the PCR by using Agarose Gel Electrophoresis.

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Experiment 5 and 6

Title: Polymerase Chain Reaction and analysis of Polymerase Chain Reaction sample.

Objectives:

  1. To amplify a 500bp fragment of lambda DNA.
  2. To understand the principles and the application of Polymerase Chain Reaction.
  3. To analyses the PCR by using Agarose Gel Electrophoresis.

Introduction:

        Polymerase Chain Reaction (PCR) is a molecular biology technique that invented by Kary R. Mullis in 1985. The PCR is a technique used to replicate a fragment of DNA so as to produce many copies of a particular DNA sequence. PCR is commonly employed as an alternative to “gene cloning as a mean of amplifying genetic material for gene sequencing, and is also used to measure gene expression. The PCR only used a small amount of DNA molecule to amplify. However, it is an in vitro technique.

        A PCR mixture consists of four key components- two primers(about 20 nucleotides long), a target DNA sequence(about 100 to 5000 base pair), a thermotable DNA polymerase(AmpliTaq polymerase, which can remain stable at 95oC or higher), and four deoxynucleotides ( dGTP,dATP,dCTP,dTTP). These components are added to a water and buffer mixture. As a little as 25 or 50 μl of this mixture may be used as a cost-cutting strategy, but 100μl is a more common amount. Volume not withstanding, the critical requirement is to maintain constant final concentrations of the reagents.

        The two strands of the DNA are separated by heating and short sequences of a single DNA strand (primers) are added, together with a supply of free nucleotides and DNA polymerase obtained from a bacterial that can withstand extreme heat. For instance, a special DNA polymerase-Taq polymerase isolated from Thermus aquaticus which is living in hot springs can withstand high temperature used to denature the template DNA. In a series of heating and cooling cycles, the DNA sequence doubles with each cycle and is thus rapidly amplified. The product generated from the PCR is analyzed by agarose gel electrophoresis.

        The PCR have three steps are repeated for 30 or 40 cycles. The cycles are done on an automated Thermal cycler, which rapidly heats and cools the test tubes containing the reaction mixture. Each step -- Denaturation (alteration of structure), annealing (joining), and extension -- takes place at a different temperature:

  1. Denaturation: At 94°C, the double-stranded DNA melts and opens into single-stranded DNA.
  2. Annealing: At 54°C, hydrogen bonds form and break between the single-stranded "primer" and the single-stranded "template." (The template provides the pattern to be copied.) The more stable bonds last longer and on that little length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
  3. Extension: At 72°C, the polymerase works best. As a result, the attraction, created by the hydrogen bonds, of the primers to the template is stronger than the forces breaking these attractions. The upshot is that bases complementary to the template are coupled to the primer.
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Materials and Methods:

  • DNA sample(λ DNA – 0.1μg/ml)
  • Primer 1:5’ GATGAGTTCGTGTCCGTACAACTGG 3’
  • Primer 2:5’ GGTTATCGAAATCAGCCACAGCGCC 3’
  • Nucleotide mix (dATP, dCTP, and dTTP – 1.25 mM each)
  • Taq DNA polymerase(5U/μl) and buffer
  • 25mM MgCl2
  • Sterile mineral oil
  • 1kb ladder (0.05 μg/μl)
  • Ethidium bromide
  • dH2O

Experiment 5

1. PCR mixture:

2. Each group need prepare one tube.

3. All the PCR components added into the small tube (PCR tube), then put into PCR machine approximately 2 hours.

4. The sample has to keep at -20oC for ...

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