Part 2: Complete Ligation Reaction Calculations

You are setting up a ligation reaction with the following components:

Plasmid Vector (0.02 µg/µL)

Insert DNA (0.03 µg/µL)

10X T4 Ligase Buffer

T4 DNA Ligase Enzyme

Water

You plan to set up one experiment with a _______ ratio of insert to plasmid. The amount of plasmid vector to be used in total will be _____ µg. The final reaction volume will be 50 µL. You will add 2.5 µL of T4 Ligase once the reaction samples are prepared (for a total volume of 52.5 µL). Complete the following chart regarding the volumes of each component used to prepare your samples.

matching

fragment DNA at specific sites

hybridize DNA fragments with labeled probe

create DNA features on a DNA microchip

prepare mRNA for DNA sequencing

transform eukaryotic cells with electricity

detect the amount of or the 5’ end of mRNA

visualize DNA, RNA or other small structures

a type of electrophoresis

detect protein binding to DNA

use antibody to detect a specific protein

use antibody to purify a specific protein

amplify a specific locus of DNA

adding transformed ES to blastocyst

Type of DNA chip that looks at DNA with modified histones

A. electroporation

B. primer extension

C. PCR

D. electron microscopy

E. Southern blot

F. immunoprecipitation

G. photolithography

H. gel mobility shift assay

I. making transgenic mice

J. restriction enzyme digestion

K. Western blot

L. PAGE

M. ChIP

N. cDNA synthesis

O. microinjection

P. spotted microarray

*Why was carbon chosen as the atomic backbone of life?

*Define a functional group and explain their importance to life.

*Describe the complete structure, classification and functions of the carbohydrates.

*List and describe in detail the 4 protein structures.

*Define an enzyme and explain how they work and their importance to living things.

*Compare the different types of triglycerides and indicate which are not healthy and those that are healthy. Also, indicate why some are and are not healthy choices.

*List all the macromolecules and describe the monomers of each and how each can be identified through testing.

*List and describe all of the accessory organs of the digestive system and explain how they assist with the process of digestion.

*Explain at least 5 ways in which the small int. is capable of increasing its surface area to perform greater absorption of nutrients.

*Describe in detail the movement of food from the mouth to the anus.

*Compare the similarities and differences between the large intestine and the large intestine.

*Compare the similarities and differences between the wall of the sm. int. and the lg. int.

An organism has the indicated traits. Based on current knowledge, indicate

whether the organism belongs to the Domain Archaea, Bacteria, Eukarya, or some

combination. Use the possible answers listed below. Place the number of the correct

answer in the space provided. An answer may be used more than once.

Possibilities:

1. Archaea 4. Could be Archaea or Bacteria

2. Bacteria 5. Could be Archaea or eukaryote

3. Eukaryote 6. Could be Bacteria or eukaryote

7. Can’t tell (could be any Domain)

_____a prokaryotic thermophile that prefers temperatures above 45°C

_____utilizes CO2 as a carbon source

_____has cell membrane lipids of branched hydrocarbons with ether-linkages

_____ has more than one type of RNA polymerizing enzyme

_____has cell walls of peptidoglycan

_____lacks membrane-bound nucleus ("nucleosome structure")

_____has mitochondria as an organelle

_____has oxygenic photosynthesis

_____ has a circular chromosome structure

_____has cell walls

Binding of an agonist to an adrenergic GPCR receptor results in:
A.   The GalphaS subunit dissociating from the Gbeta-Ggamma subunits.
B.   The Gbeta subunit dissociating from the GalphaS-Ggamma subunits.
C.   The Ggamma-subunit dissociating from the GalphaS-Gbeta subunits.
D.   None of the above are correct.
E.   All of the above are correct.

mTORC signaling occurs when:
A.   the cell has an energy deficit and requires more nutrients.
B.   the cell has an adequate supply of nutrients and is in a high-energy state
C.   the muscles are fatigued and require additional oxygen.

In the citric acid cycle, the acetyl-coA is linked to a pre-existing molecule of_______________.
A.   citrate (citric acid)
B.   oxaloacetate
C.   fumarate
D.   succinate
E.   pyruvate

What type of enzyme is required to catabolize an unsaturated fatty acid but not a saturated fatty acid?
dehydrogenase
mutase
hydratase  
isomerase

please answer all of it or it cannot don't answer question

1) Think about the bisubstrate reaction F + G -> H that can be catalyzed by an enzyme that 10) obeys Michaelis-Menten kinetics. Assume that [G] is so high that it is essentially unlimited.

Which one of the following scenarios is the one that can best be described as "first order with respect to F"?

=> [F] is three times lower than the Km.

Explain Why answer is [F] is three times lower than the Km.

14. Choose the answer and Explain Why
    If no ESI complex is ever formed, we can best conclude, based on our coverage of reversible 1inhibitors, that:

    1. A) the inhibitor is an uncompetitive inhibitor (but not the other two types)

    2. B) the inhibitor is a noncompetitive inhibitor (but not the other two types)

    3. C) the inhibitor could be either an uncompetitive or a noncompetitive inhibitor (but not the other type)

    4. D) the inhibitor is a competitive inhibitor (but not the other two types)

    5. E) the inhibitor could be either a competitive or an uncompetitive inhibitor (but not the other

       type)

3. Assume that a bacterial culture is grown on medium containing radioactive thymine until essentially all of the thymine in the DNA is radioactive. Then the bacteria with labeled T were washed and transferred to a medium containing nonradioactive T for one round of replication. Given the four double stranded sequences below, which one is likely to represent the replicated double stranded structure? Note: A,C,T,G represent typical nucleotides, and 3T represents labeled T.)

In PCR reactions, the only enzyme that is needed is a polymerase. Provide reasoning as to why the following essential replication enzymes/components are not needed:

Helicase

Topoisomerase

Ligase

Primase

Replication clamp

Due to system limitations you are attempting to clone a small bacterial gene using a poorly characterised bacterial plasmid. You know that the plasmid contains only two drug resistance markers (one for resistance to tetracycline, another for resistance to ampicillin). You have determined that the restriction enzymes EcoRI and BamHI each cut the plasmid only once while SauIII and RsaI cut the plasmid twice. You also have determined that the EcoRI and SauIII recognition sites are within the tetracycline resistance marker, but the BamHI and RsaI recognition sites are not within or even close to either drug resistance marker. You must use this particular vector: a. Which restriction enzyme(s) should you use and why? b. What will happen if you use the BamHI recognition site? What will happen if you use the RsaI recognition site? (Describe this with regards to the functioning of the drug resistance marker and which antibiotic(s) you should use in the media)

1. Regarding countercurrent mechanisms:
   1. List which structure(s) is/are responsible for establishing the concentration gradient in the renal medulla? Describe how this is accomplished. (5 points)
      1. Note: You do not need to describe the entire positive feedback loop, I am looking more simply for structures or structural modifications and functions of those structures. Do mention how the tubular fluid concentration changes and the interstitial fluid of the medulla is affected as a result.
   2. List which structure(s) is/are responsible for maintaining the concentration gradient in the renal medulla? Describe how this is accomplished. (2 points)
   3. ACE inhibitors (angiotensin converting enzyme inhibitors) are a popular class of drug that treats hypertension. Use your knowledge of the renin-angiotensin-aldosterone system to describe the physiological mechanism of how this medication works at the nephron and kidney level to reduce blood pressure. (3 points)
   4. Predict how ADH hypersecretion would affect the sodium concentration of the urine, and explain why. (2 points)

12) Imagine a pulmonary embolus breaking up and entering the pulmonary artery supplying blood to an entire lobe of the left lung, blocking 75% of the pulmonary arterioles in that lobe. Describe the effects on the arterioles and bronchioles in this lobe in response to this event.

13) Elevated potassium levels in the body (hyperkalemia) is a serious condition, resulting in a severely decreased ability for muscle fibers and neurons to reploarize. The adrenal cortex can respond to this. However, very high osmolarity shuts down this response. Explain this incongruity, by describing the response mechanism of the adrenal cortex. (6 pts)

16) ) A vaccine for Coronavirus is likely to take a longer time than imagined, because the most obvious target of the vaccine should be the external spike protein on the envelope of this enveloped virus. The cellular attachment protein on epithelial cells, which the virus binds to for entry into the epithelial cell, is known to be ACE2, a receptor enzyme. Explain why the production of a vaccine targeting the coronavirus spike protein (S1), is not likely to be successful.