Lab 7 Flashcards
What would happen if you did not use a chelating agent such as the InstaGene matrix?
Chelating agents are required because they remove positively charged ions from a solution. Some enzymes require these cations to function. therefore, without the cations, the enzyme will not function; thus, the DNA will remain intact. If the DNA was degraded, there would be nothing for the PCR tone performed on.
What is needed from the cells for PCR?
The only thing needed for PCR from cells is DNA. All other required elements are added later into the reaction tube.
What Structures must be broken to release DNA from a cell?
the cell membrane and nucleus must be broken.
Why is it necessary to have a primer on each side of the DNA segment to be amplified?
this is the only way to get a segment at the proper length.
How did Taq DNA polymerase acquire its name?
comes from the microbe Thermus Aquaticus, a microorganism that has an optimal polymerase temperature between 75-80 degrees Celsius.
Why are there nucleotides (A, T, G, and C) in the master mix? what are the other components and their function?
nucleotides are in the master mix as hey make up DNA, the monomers that get joined together to build a DNA strand. Therefore, they are used to synthesize the new copied strands of DNA. Also in the master mix is the Taq polymerase, Mg 2+, the DNA template and the primers.
Describe the three main steps of each cycle of PCR amplification and what reactions occur at each temperature.
i. denaturation- high temp (~94 C) is used to melt (denature) the DNA strands. DNA goes from double to single strand.
ii. Annealing- Typically occurs ~60 C. This is the point when the primers hybridize (bind) to their targeted (complementary) region on DNA. Because there are so many primers and they are much smaller than the DNA molecules, they have a better chance of annealing to their targeted region, compared to the two strands of DNA coming together again.
iii. Elongation- Done at 72 C. this is when new DNA strands are synthesized. Taq polymerase works best around this temp.
List the three possible Alu genotypes at locus PV92.
i. Homozygous for the insert (+/+)
ii. Heterozygous (+/-)
iii. Homozygous for no insert (-/-)
Why is it necessary to use PCR? Why can’t we extract DNA and immediately put it on the gel?
PCR is required because the chromosomal DNA is not at a high enough concentration to be visualized on a gel. we can only see the DNA as all the same sized fragments migrate to the same distance.
What does ethidium bromide do?
in the gel it intercalates between the bases of the DNA fragments, and when the gel is exposed to UV light it is the concentrated DNA- bound ethiduim bromide that forms the band.
What is agarose?
a highly purified form of agar ( a polysaccharide extracted from the cell walls of a certain algae)
How is agarose made?
by weighing a particular amount of agarose and boiling it in a buffer. the gel is poured onto a plate and a comb is inserted.
what determines the pore size I the gel?
the more agarose in the gel the smaller the holes.
does the DNA move toward the positive or negative electrode and why?
positive electrode because the negative phosphate groups in the DNA backbone
