Question

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Why is it necessary to chelate the metal ions from solution during the boiling/lysis step at 100C? What would happen if you did not use a chelating agent such as the InstaGene matrix? 1. 2. What is needed from the cells for PCR? 3. What structures must be broken to release DNA from the cell? Why is it necessary to have a primer on each side of the DNA segment to be amplified? 4. 5. How did Ta? DNA polymerase acquire its name? 6. Why are there nucleotides in the master mix? What are the other components of the master mix, and what are their functions? 7. Describe the three main steps of each cycle of PCR amplification and what reactions occur at each temperature.

Answer 1

1) It necessary to chelate the metal ions from solution during the boiling/lysis step at 100°c to remove the Magnesium because they act like cofactors, removing this inactivates the DNases, which then keep the DNA template intact.

If we don't use the e chelating agent, then it will not grab the metal ions out of the solution therefor DNases would remain activated and would degrade the DNA, resulting in NO PCR AMPLIFICATION.

2) The genomic DNA released from the nuclei of the cells is needed

3) The cell and nuclear membranes

4) So that both of the sides of the double helix are used as templates

5) Taq polymerase is a thermostable DNA polymerase. It is named after the thermophilic bacterium "Thermus aquaticus" from which it was originally isolated. Its name is often abbreviated to Taq Pol or simply Taq.