Understand how a standard curve is constructed for DNA and protein gels. How do
ID: 87731 • Letter: U
Question
Understand how a standard curve is constructed for DNA and protein gels. How do you measure the bands? Know what is on the x-axis and y-axis, how to draw the line and how to determine information on sizes of unknown DNA fragments and proteins if you are given a hand drawn plot (no equation). These are always semi-log plots. What is the limitation of any such standard plot? Why do you need to create one for every gel? Why can’t you just use the same one over and over?
Understand how to use an absorbance vs. protein concentration standard curve to determine protein concentration of unknown samples. Know the limitations of this type of plot (i.e., when you must dilute your sample further to get an accurate concentration). Why do you need a blank?
Understand how LCT/lct is inherited, what phenotype and genotype mean, and how to determine the genotype from comparison to predicted PCR products for each allele. Where is the mutation that we analyzed? What is the effect of this mutation on the lactase gene? In more general terms, how do you determine a predicted phenotype from the genotype?
Consider how the development of domestic herds (cattle, sheep, goats) impacted the LP/LNP phenotypes. Why was there an increase in LP in these herding cultures? This was a random mutation – why did it become prevalent?
Understand how PCR can specifically amplify a selected region of DNA for analysis. Why do you need primer pairs? How does this take advantage of the requirements of DNA replication? Recall that replication cannot begin without a primer – so PCR takes advantage of this fact to direct replication to a desired region. What is special about Taq DNA polymerase that makes PCR possible?
Understand the steps we used to purify the DNA for PCR. Why were the samples boiled? Why was it necessary to centrifuge the sample after this? What was the purpose of the Chelex? Was the DNA in the supernatant or the pellet?
What is apoptosis and how does it differ from necrosis? Why does the DNA present a “ladder” of equal “steps” (size differences)? What caused this to occur? What did you see in the control lanes? What do you expect for the size of the control DNA? What would a gel with necrotic DNA look like? Be sure to consider the role of nucleosomes and linker DNA in this process.
What is IgG? What is the structure of IgG? How much IgG is typically found in serum (This can be found on the Powerpoint). How does this compare with the yield you obtained (from 1 ml of dialyzed serum) by the purification procedures we used in lab? Why do you suppose our yield is not close to 100%? Where might we have ‘lost’ protein? What is the advantage of beginning our purification of IgG with sheep serum rather than sheep blood? What did dialysis accomplish? Was this a purification step? Why or why not?
What is ion-exchange chromatography (IEC)? How does it work? What types of proteins were removed at this step? Why wasn’t IgG removed as well?
What size is native IgG? How did we use A595 and Bradford reagent to track the purification process? What information did we get from this (Does it tell you how much IgG is present)?
How do you decide whether or not you need to dilute a protein sample? How do you dilute a sample 1:10? 1:100? You can think about this in general terms, even without knowing a specific volume. Then consider a) if you want a final volume of no more than 1 ml; b) if you are asked to start with a volume of no less than 10 µl. c) if you are told to make exactly 500 ul of a diluted sample.
Given a sample with a protein concentration of 40 mg/ml how would you dilute it to get a concentration of 10 µg/µl? 1 µg/µl? Could you dilute it to 100µg/µl? Why or why not? Again, you can think about this without specifying a starting or final volume – but then consider if you want 100 µl final. Can it be done in each case?
Finally, you are loading protein samples on a gel. Your starting concentration is 80 mg/ml. You can load no more than 10 µl on the gel, and want to load 15 µg of protein. How would prepare this sample? (Note: this does NOT mean that you MUST make only 10 µl.) What if your starting sample was 200 mg/ml? Remember, you are limited by the set of pipettes available in the lab.
Why did we need to run the SDS gel to determine if the purification had worked? What did you expect to see after each step? Why was it necessary to treat the proteins with SDS and dithiothreitol (DTT) before loading them on the gel? How did this alter the appearance of the IgG? Could you look at a series of samples run on a protein gel and put them in relative order of purification (assuming you had a control lane with the pure protein you were trying to isolate)? Why did we want to have about the same amount of total protein in each lane of the gel?
Bioinformatics background info: What is meant by antibiotic resistance and why is it such a significant problem? What are some causes of antibiotic resistance? What are common targets in prokaryotes of antibiotics? What is bioinformatics and how can it help us to understand antibiotic resistance?
How can a mutation lead to antibiotic resistance? Why might some changes in the DNA sequence not have an effect on either the sequence of the protein or the ability of the bacterium to resist the antibiotic? Why might some changes in the protein sequence not have an effect on antibiotic resistance?
When translating a sequence of DNA, what online tool might you use? When translating a double stranded DNA in the “TRANSLATE” program from EXPASY, why does the site return 6 different protein sequences from a single DNA sequence? How do you determine which protein sequence is the correct one to use for your analysis?
Both CLUSTAL and BLAST gave you information about similarity between sequences. When would you want to use CLUSTAL over BLAST and vice versa?
Be able to explain the roles of GA and ABA in dormant and germinating seeds. What is the stimulus that causes the seeds to transition from dormancy to germination? The 3 main tissue types in the seed are the embryo, endosperm, and aleurone. Briefly describe the function of each. Where is the ‘source’ of GA in the seed? What is its ‘target’ tissue (be specific)? How did we assay for a positive response to the hormone signal? (i.e. what did we measure and why)?
Be able to interpret your lab group’s data and draw conclusions. If you were shown a graph of one of the experiments, could you describe what is represented and explain why it occurred? Looking at your data, why do you suppose it took more than 24 hours to detect a response beyond the baseline value observed at T=0? At what point did you see the maximum amount of starch digestion (above the 0 hr imbibition value)? Did the addition of extra GA, ABA, or both for 48 hrs alter the amount of starch digestion? If so, in what way?
Be able to explain why the endosperm half-seeds that imbibed water for 72 hours have little-to-no induced amylase activity (above the baseline amount seen in 0 hr imbibition) and why it increased when GA was added to the water. Why do you suppose that the endosperm half-seeds + GA had greater amylase activity than the embryo half-seeds +GA?
Explanation / Answer
As per Chegg guidelines we are supposed to answer one question only. So, I'll answer only one question out of these 21 questions.
PCR: polymerase chain reaction
PCR is used for the amplification of DNA you can understand that as making multiple copies of DNA.
PCR can amplify desired region on DNA as the primers we use are oligonucleotides that are complemantory to the target region. The primers designed for human specific DNA will only bind to the target region of human DNA only. That is how we can amplify the desired region.
We use two pair of primers one is forward primer and another is reverse primer. We use two set of primers so that copies of both the strands of the double-stranded DNA can be made simultaneously. Both the primers will bind in the 5 prime to 3 prime direction of the DNA.
As in replication denaturation step and the RNA primers are attached after which the nucleotides binds and DNA is replicated. Same way in PCR the denaturation step and primers are there.
Firstly the denaturation of DNA strands around 95 degree celsius takes place. The two strands were separated and then the annealing step takes place in which the designed primers are bound which attach to the opposite strands at 5 prime to 3 prime direction, the temperature at which primers bind is 55-65 degree celsius. Then the extension takes place under the presence of DNA polymerase at around 72 degree celsius as polymerase facilitates binding of dNTPs . The reaction mixture also has buffer and DNTPS which were the nucleotides(dATP, dCTP, dGTP, dTTP).
The role of taq polymerase is that the DNA polymerase we use in PCR is taq polymerase. It is an enzyme extracted from Thermus aquaticus. Taq polymerase can withstand High temperature and can maintain its structure and Function even at high temperature. Because it is isolated from bacteria that are evolved to survive in hot environments. So taq polymerase will be able to work properly in these shifts of temperature from 95 to 55-65 to 72 and again 95 and so on.
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