1. Why is it necessary to chelate the metal ions from solution during the boilin
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Question
1. 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? 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 Taq 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 temperatureExplanation / Answer
Answer 1. It is necessary to chelate the ions because this process will help in grabbing the enzyme cofactors which disable the enzymes that have the ability to degrade DNA. The degrading enzymes are released along with the DNA at the lysis step. If the cofactors are not present, the DNA would be degraded.
Answer 2. A sample of DNA which contains the DNA sequence of interest. This sequence should be intact and not broken.
Answer 3. The plasma membrane and nuclear membrane.
Answer 4. The primers are complementary to sequences of flanking region of the gene of interest to be amplified. Primers determine what segment of DNA will be amplified, because during each PCR cycle the DNA polymerase binds to, and extends each primer from its 3' end, generating newly synthesized strands.
Answer 5. The Taq DNA polymerase acquired its name from the organism it is isolated i.e. Thermus aquaticus.
Answer 6. There are nucleotides in the master mix because they are used form the strands complementary to the existing strands of DNA during PCR as the DNA is made up of nucleotides. The other components of the master mix are the forward and reverse primers which identify the DNA replication initiation site, the DNA polymerase which extends the primers and assembles the nucleotides is also present in the master mix. A salt buffer which maintains a neutral pH for the PCR reaction to occur; and mg2+ which is a cofactor required by DNA polymerase.
Answer 7. The three main steps of each cycle of PCR amplification and the reactions and temperatures at which they occur:
Step one is the Denaturation of DNA strands which occurs at 94oC for 1 min. Heating of strands to this temperature allows for the breaking of the hydrogen bonds and as a result, the two DNA strands separate.
Step two is the Annealing of forward and reverse primers to the corresponding strand which occurs at 60oC for 1 min. The reaction is cooled from 94oC to 60oC in this step to allow the primers to anneal to the complementary regions on each strand, thereby flanking the region to be amplified.
Step three is the Extension of DNA strands by an enzyme known as DNA polymerase. This step occurs at 72oC for 2 min. Most commonly, Taq polymerase is used to attach to the primers and extend each primer from its 3' end, generating newly synthesized strands.
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