LS7L Final - PFLUEGL UCLA

In the B-galactosidase lab, why was o-nitrophenol-B-D-galactosidase (ONPG) used?

It is a derivative of lactose, used as a chromogenic substrate to assay B-galactosidase activity.

Why is it important to know the cell density at both the 20 minute and 70 minute time points? How would NOT knowing the cell density affect your calculation of the Miller units?

We want to understand that including the cell density readings allow us to normalize by cell density. This is important because the cells are actively growing during this experiment, so there will be more cells present at the 70 minute time point than there are at the 20 minute time point. If you didn't have the cell density readings you might think that the increase in b-gal activity at the 70 minute time point is due to increased number of cells (this is NOT TRUE, since we do normalize for cell density!)

Based on your data, is the level of b-galactosidase activity influenced by the type of sugar present in the growth medium? Explain using your results.

Yes, you should be able to see that b-gal activity is induced by lactose but not by glucose.

For the tube(s) that had high levels of enzymatic activity, did you see a high level of activity at the 20 minute time point, the 70 minute time point, or both? Why might this be the case?

You should only see a high level of activity at the 70 minute time point. This is NOT due to increase in cells (see Q1). The difference in b-galactosidase activity between 20 and 70 minutes reflects the time that it takes to induce b-galactosidase synthesis after exposure to lactose.

In what direction is the reverse primer ("R") conventionally read?

5' to 3'

Which strand of the DNA (top or bottom) will the forward primer ("F") hybridize to during the PCR reaction?

Bottom

Will the DNA synthesized starting from the forward primer be the same sequence as the top strand or the bottom strand?

Top

Will the DNA synthesized starting from the reverse primer be the same sequence as the top strand or the bottom strand?

Bottom

Why would the sequence of the control region of mtDNA be more variable than a protein coding region?

In the coding region there is less tolerance for mutations, since a mutation can change protein function and have a negative impact, so tends to be selected against. The control regions contain binding sites for various proteins separated by generally unimportant regions and can tolerate many changes without altering their overall function as control regions.

Do you think this is a good primer pair? Why or why not? (Review Table 1 for some guidelines)

Answers will vary! They should take into account all the factors in the table above (question 14). In this example the primers seem okay. Length, GC content and melting temp are all in range, and Tm is similar for both primers. The left primer has a long run of bases which isn't great, and neither primer has a GC clamp on the 3' end.

Are the melting temperatures/length of your two primers similar or different? How could the melting temperature/length affect your PCR amplification?

Answers will vary! Having different melting temperatures can impact PCR because it's more difficult to choose temperatures for your PCR cycles that will work for both primers. Length also affects Tm, so having very different lengths usually results in different Tm.

Do you think this is a good universal primer (able to amplify all haplogroups)? Why or why not?

Answers will vary! But if they are amplifying a relatively short region of DNA that specifically surrounds the mutations for their haplogroups, then it probably isn't great universally. But if they're amplifying a fairly large chunk of the region, then it might work well. If the 3' end of the primer falls on a variable site, that makes it less likely to be a good universal primer.

Blood

Transports nutrients, oxygen and hormones. Regulates acid-base balance and body temp. White blood cells fight infection, platelets help blood clots to seal leaks.

Bone

Provides protection, forms red blood cells, stores calcium and phosphate, aids movements (by serving as levers for muscles)

Liver

Detoxification of metabolic waste, storage and release of energy (glycogen), destruction of old red blood cells, synthesis and secretion of bile

Lung

Filters, humidifies and adjusts air temp, primary site of gas exchange

Neurons

respond to stimuli, conduct signals to coordinate responses

Skeletal muscle

contract to allow voluntary body movement

Skin

heat regulation, protection, excretion (sweat), aids in synthesis of vitamin D, sensation

Aorta

deliver oxygen and nutrients, remove CO2, carry blood away from the heart

Did you compare your two groups using the mean or the median as the summary measure? Please give a reasoning for your choice.

We decided to use the median since neither of the datasets has a normal distribution, and the mean is not recommended unless all data is normally distributed.

Considering the p-value, the observed difference between your summary measure, and the datasets - what are your overall conclusions regarding your experiment?

Individuals who are shorter (5'0" - 5'6") have a higher maximum heart rate after exposure to cold stimulus than individuals who are taller (5'6" - 6'0"). The observed difference of the medians was about 4 beats per minute. There is a lot of individual variation, with some suspected and confirmed outliers indicating a wide range of maximum heart rates within the subject pool.

How could you use electrophoresis to determine if the subunits of an unknown protein are associated by disulfide bonds? Be sure to describe the possible results. Limit your answer to 5 sentences.

Run the protein twice - once with SDS alone and once with SDS and a reducing agent. If the subunits are associated by disulfide bonds and are different sizes, more bands will be observed in the presence of the SDS and the reducing agent than with SDS alone. If the subunits are associated by disulphide bonds and are the same size, then the number of bands will be the same in the presence of the reducing agent and SDS alone.

Last week you boiled your cheek cells in order to isolate your DNA. From what you know about protein structure, would the proteins released by boiling your cheek cells be more likely to stay in solution or precipitate out of solution? Please explain and limit your answer to 3 sentences.

Heating proteins generally results in unfolding of the polypeptide chain. This frequently leads to aggregation of the denatured proteins and the precipitation out of solution.