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You are doing a mini prep, and you're performing every step exactly to instructions. You are on the drying step. After drying your column you want to keep

a. the column and the collection tube. Discard the flow through.

b. the column. The collection tube and flow though are waste.

c. the column, collection tube and the flow through.

d. the collection tube and flow though of the column discarding the column.

e. nothing. Discard everything.

f. the flow though, putting it into a new tube.

Only one will be correct. Explain

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27 Aug 2023

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Related questions

can anyone paraphrase the following procedure for me please. I prefer to be typed because the handwriting sometimes is not clear

First experiment: Bacterial Transformation and Affinity Purification of Protein:

Transformation procedure:

Bring 2 test tubes and label them +pGLO and –pGLO,

Transfer 250 ul of transformation solution to each tube and then place them in ice.

Pick a single colony of bacteria using a sterile loop from the bacterial plate and immerse the loop in +PGLO tube and start spinning the loop to mix, repeat the same step for –pGLO and place both tubes in ice again.

Then, immerse a new sterile loop in the pGLO DNA plasmid stock tube and withdraw a loopful of the plasmid and immerse it in the +pGLO tube to load it and place the tube in ice again. No pGLO plasmid will be added to the –pGLO sample as labeled.

Incubate the tubes in ice for 10 minutes and take the following labeled plates from the instructor: LB/amp + pGLO, LB/amp/ara + pGLO, LB/amp – pGLO, and LB – pGLO. There will be a total of four plates.

Heat hock will start by transforming both tubes to 42C water bath for 50 second and then returning them to ice again.

Add 250 ul of LB nutrient broth to both tubes and incubate at room temperature for 10 minutes.

Using a new sterile pipet for each plate, transfer 100 ul of the –pGLO solution to the, LB/amp – pGLO and LB – pGLO agar plates and 100 ul of +pGLO solution to the LB/amp + pGLO, and LB/amp/ara + pGLO plates.

Spread the suspension using sterile loops and place your plates upside down in the 37C incubator overnight.

Bacterial growth will occur and result can be analyzed the day after.

* For data analysis, observe the result in the plates under normal light conditions and then under UV light and right your observations. This will include observing the number of colonies, colonies color and growth. Transformed bacteria will look green in color under UV with the presence of arabinose sugar in the media and will look white in color if arabinose is absent. No growth will be shown if the bacteria are not transformed.

Second experiment: Affinity purification of green fluorescent protein:

Affinity purification procedure:

Re-observe you plates from previous experiment under natural light and UV light. Identify several green colonies on the LB/amp/ara plate and with the use of a loop scoop up some cells and immerse the loop in a new culture tube labeled (+), the same procedure is done for the LB/amp plate in a tube labeled (-). Disperse the colonies in the culture tubes and shake vigorously. And place in an incubator for 24 hours.

Transfer the entire culture to a 2-milliliter microtube labeled +. Centrifuge for 5 minutes and remove the supernatant.

Re suspend the pellet in 250 ul of TE solution and then add 1 drop of lysozyme and mix then place the tube in the freezer to help rupture the cells.

For bacterial lysis, thaw the microtube and centrifuge for 10 minutes at maximum speed. And prepare the chromatography column.

Before performing the chromatography, add 2 milliliters of Equilibration Buffer to the column, and Drain the buffer from the column until it reaches the 1 milliliter.

After centrifugation, transfer 250 μl of the supernatant into the new microtube and transfer 250 μl of Binding Buffer to the microtube and Place in the refrigerator.

Label 3 collection tubes 1, 2 and 3. Load 250 μl of the supernatant in Binding Buffer into the top of the column and Let the entire volume of supernatant flow into tube 1.

Transfer the column to collection tube 2. Add 250 μl of Wash Buffer and let the entire volume flow into tube 2.

Transfer the column to tube 3. Add 750 μl of TE buffer and let the entire volume flow into tube 3.

Examine all collection tubes using the UV lamp and note any differences in color between the tubes.

Biochemistry Protocols: Your First Protein Purification

As the newest student in a biochemistry research lab, you are given responsibility for purifying a mitochondria protein. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answers.

(a) You pick up 20 kg of beef hearts from a nearby slaughterhouse. You transport the hearts on ice, and perform each step of the purification on ice or in a walk-in cold room. You homogenize the beef heart tissue in a high-speed blender in a medium containing 0.2 M sucrose, buffered to a pH of 7.2.

Why do you use beef heart tissue, and in such large quantity?

What is the purpose of keeping the tissue cold and suspending it in 0.2 M sucrose, at pH 7.2? What happens to the tissue when it is homogenized?

(b) You subject the resulting heart homogenate, which is dense and opaque, to a series of differential centrifugation steps. What does this accomplish?

(c) You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria. Next you osmotically lyse the mitochondria. The lysate, which is less dense than the homogenate, but still opaque, consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate you add ammonium sulfate, You centrifuge the solution, decant the supernatant, and discard the

pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample, but this time you save the pellet because it contains

the protein of interest. What is the rationale for the two-step addition of the salt?

(d) You solubilize the ammonium sulfate pellet containing the mitochondrial proteins and dialyze it overnight against large volumes of buffered (pH 7.2) solution. Why isn’t ammonium sulfate included in the dialysis buffer? Why do

you use the buffer solution instead of water?

(e) You run the dialyzed solution over a size-exclusion chromatographic column. Following the protocol, you collect the first protein fraction that exits the column, and discard the rest of the fractions that elute from the column later. You detect the protein by measuring UV absorbance (at 280 nm) in the fractions. What does the instruction to collect the first fraction tell you about the protein? Why is UV absorbance at 280 nm a good way to monitor for the presence

of protein in the eluted fractions?

(f) You place the fraction collected in (e) on a cation exchange chromatographic column. After discarding the initial solution that exits the column, you add a washing solution of higher pH to the column and collect the protein fraction that immediately elutes. Explain what you are doing.

(g) You run a small sample of your fraction, now very reduced in volume and quite clear (though tinged pink), on an isoelectric focusing gel. When stained, the gel shows three sharp bands. According to the protocol, the protein of interest is the one with the pI of 5.6, but you decide to do one more assay of the protein’s purity. You cut out the pI 5.6 band

and subject it to SDS polyacrylamide gel electrophoresis. The protein resolves as a single band. Why were you unconvinced of the purity of the “single” protein band on your

isoelectric focusing gel? What did the results of the SDS gel tell you? Why is it important to do the SDS gel electrophoresis after the isoelectric focusing.

Check the Course Announcements at the beginning of the week following the deadline for Part 1 to discover exactly what happened to the deer population on the Kaibab plateau. This information is essential for completion of Part 2.

The second part of your project is to take what you've learned about the Kaibab forest ecosystem and write an accurate description of the boom and bust of this deer population, using correct ecological language. Consider as many of the aspects of these events as you can. Be sure to consider the effects on the other species in the ecosystem as well.

Refer to your on-line materials and the appropriate sections of your textbook. Your discussion should include (but not be confined to) consideration of all of the following concepts: carrying capacity, predator/prey relationships, population cycling, resource partitioning, competitive exclusion, fundamental and realized niches, food chains, food webs and trophic pyramids.

Note that the purpose of this exercise is for you to demonstrate to me that you understand these important concepts of ecology by utilizing them accurately and appropriately in the description of the disaster in the Kaibab forest. Your essay must include both clear indications that you understand the meanings of these principles and clear indication that you understand how these principles can be used to describe what happened to the ecosystem on the Kaibab plateau.

information given of part one: On the north rim of the Grand Canyon, an area called the Kaibab plateau, there is a conifer forest called the Kaibab forest. The forest's trees include pine, fir and spruce. Shrubbery includes a variety of willows, raspberries, etc. A hundred years ago, this forest was a stable ecosystem, housing, among other species, a population of about four thousand mule deer.

These deer were hunted by local Native Americans, who used them for food and other materials. They were also hunted by predators like pumas, wolves and coyotes.

In 1906, the US made this region into a National Game Preserve. With the intent of protecting the deer, hunting was forbidden. Bounties were paid for killing the pumas, wolves and other large animals that preyed on the deer. By 1925, more than 6000 predators had been killed, virtually wiping out the ecosystem's large predators.

*Read the instructions* for Part 2, then consider this additional information about the Kaibab forest.
There are some hints in here to suggest the *minimum* range of the subjects you need to discuss to explain this; You will need to read the chapters and the course materials to understand the meanings of these concepts, and to be able to use them to explain the Kaibab disaster. There are other important concepts as well--so keep your eyes open as you read. It wouldn't hurt to go back and read the original description in Part 1, either. And you may find it necessary to do some quick browsing in areas of your book not directly related to ecology. (For instance, you might just want to do some looking into the nature and importance of the bark and twig ends on a tree.) Do not neglect the on-line essays which are provided for you in this Unit. What you should *not* do is Google the Kaibab, or look it up on a source like Wikipedia. This is not about what you can *discover* about the Kaibab forest.
Your grade will depend upon how completely you convince me you understand the concepts involved in this description. This is your test on ecology--your goal is to convince me that you understand the concepts and terminology of the subject, and can apply them to a real life situation.
Before we go on to the new information, there are a few things that came up as I was grading the Part 1 submissions which I think I should mention, just to keep you from straying in some incorrect directions.
First, remember that your concern is the fate of the Kaibab forest in the *immediate* sense--over tens of years, not thousands. There are no evolutionary issues here, at least in the short term (and that's your concern right now). So we won't be talking about adaptive radiation.
Also, keep your focus on the *Kaibab forest*, not on other ecosystems. Oh, and remember that the Kaibab is pretty isolated--emigration really isn't a reasonable alternative for the species living in this forest.
And finally, until we get to Part 3, the only human interaction we are considering is the initial decision to put bounties on those large predators, which led to their near-extinction in the area. You will be thinking a bit about human hunters trying to control the deer population toward the end, but the rest of the time you're just concerned with the natural processes going on in the ecosystem.
Okay, now on to the actual description of what happened in the Kaibab Forest as a result of killing almost all of the large predators. Remember, your task in Part 2 is going to be to describe and explain all of this using the concepts of ecology which you are learning in Unit 2 of the course.
*****
The primary, most immediate outcome of the removal of the predators from the ecosystem was that the deer population exploded, because the loss of the normal predator populations removed a primary source of population control. And the expanding deer population ate a *lot.* [Hint: explaining this requires discussion of population cycling, predator prey relationships and carrying capacities.]
As the deer population got larger and larger, their impact upon their favored food plants became extreme. They ate a much larger percentage of each year's plant populations, and they ate a larger portion of each individual plant. They also began to eat more from the plants which they didn't like so well--plants normally consumed by other herbivores in the ecosystem. In the winter, the starving deer ate about anything remotely resembling food, including twigs and bark from trees. As a result of the destruction of many of the twig ends of the trees, the spring tree growth was inhibited (why?). The stripping of the bark caused the death of some of the trees (why?). The extreme browsing on the herbaceous plants caused shifts in the plant population diversity, as the deer drove many of their favored plants into extinction within the ecosystem. [Hint: Explanation here will need to include discussion of resource partitioning, competitive exclusion, and realized vs. fundamental niches.]
All of this also impacted upon the rest of the animals in the ecosystem, since many of them depended upon the same kinds of food the deer ate, or depended upon the foods that the deer were eating because they could no longer get the food they favored. Then of course, the smaller predatory species (like foxes and owls) depended upon the health of those populations of small herbivores (like rabbits and mice). [Hint: sounds like food chains, food webs and trophic pyramids come into this part.]
After a couple of decades, the ecosystem was quite altered. The plant community was very different from what it had been before, and the deer herd, though not extinct, was composed of significantly smaller and weaker deer than the herd from the 1890s. (This was partially due to their straightened circumstances re. food, but also partially due to the absence of predators. Why would the removal of the natural predators cause this change in the general vigor of the deer in the herd (and why would the substitution of *human* hunting not exert the same positive influence)?)
Okay, the next phase of your assignment is to describe all the things going on in this ecosystem using the proper terminology that you should be learning from studying this unit of the course. Note that your use of terms like, for instance, "resource partitioning" needs to show that you understand not just what the concept means, but how it is reflected in this specific ecological disaster. Feel free to discuss this as much as you want to on your Discussion Board. Don't ignore the little questions tucked into the above description, and pay attention to those hints. Your response must demonstrate proper understanding of these concepts and how they are demonstrated by the disaster in the Kaibab forest.
This is the largest part of your exam project; your response should reflect the fact that this is a 50 point "essay" question. You are given a lot of organizational clues and hints about what issues you need to include in the information above. Make use of those things.

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