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1.You need to do a transfection of your tissue culture cells, but before you do,

you must so some prep work. Today, you have to plate your cells equally in a

6-well plate. You have to first count your cells. You trypsinize your cells and

add media to a total volume of 5mLs. You remove 20µL of those cells to a

1.5mL tube with another 20µL trypan blue.

a. You count the following live cells in the four quadrants:

i. Quadrant 1 – 168

ii. Quadrant 2 – 132

iii. Quadrant 3 – 197

iv. Quadrant 4 – 117

How many cells/mL do you have?

How many mL will you need to plate 500,000 cells into one well? Add media to 2mL of total volume.

2.Once you plate your cells, the next step is to prepare the DNA for the transfection. You read your DNA on the spectrophotometer and it tells you that you have 12µg/mL of DNA, with an A260 of .413 and an A280 of .22

a.What is your purity

b.Is this a good purity for downstream use in transfection or PCR? Provide the recommended purities for RNA and DNA.

c.What is one reason a sample could have a bad purity after isolation/purification?

3.Walk me step by step how to pour 1% gels from scratch. 2 gels = 100mL

a.Make 1L of 1x TAE from a 50x stock

b.Weigh out the agarose for 2 gels

c.How much TAE to add?

d.Heat up obviously

e.Cool down

f.Add Ethidium Bromide, how much?

i.Also note a safety precaution

g.How much DNA do I load and how much dye to add?

h.What happens if there is no dye?

i.What voltage and time do you set?

3.Make a buffer from scratch

a.1L of 5M NaCl and 1L of 1M Tris (look up MW)

b.Once your stock solutions are made make 1L of 200mM NaCl and 20mM Tris (use water to fill to 1L)

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Keith Leannon
Keith LeannonLv2
28 Sep 2019

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Related questions

2. After your frustration with tissue culture, you finally get your cells passaged and decide to set up your cDNA synthesis reaction, PCR, and agarose gel. You have extracted RNA from your cells, and now you need to proceed with the cDNA synthesis.

a. The first step is to determine the concentration of your RNA. You dilute your RNA 1:250, vortex it, move it to the cuvette, and run it on the spectrophotometer. The spec tells you that your concentration of RNA is 22µg/mL with an A260 of 0.32 and an A280 of 0.2. What is your ACTUAL RNA concentration, and what is its purity?

b. After determining your actual concentration of RNA, you are ready to make your cDNA. The protocol calls for the following for a total of 20µL:

5x iScript Reaction Mix – 4µL

iScript reverse transcriptase – 1µL

Nuclease free water – x µL

RNA template (100fg to 1ug total RNA) – x µL

You decide to use 500ng (or 0.5µg) of RNA in this reaction. How many µL of your RNA do you need, and how much water will you put in the tube?

c. You make the cDNA and get ready to set up your PCR for CD-10. The Qiagen protocol is as follows to a total of 50µL:

cDNA – 2µL

MgCl (25mM) – 4µL

CD-10 Forward Primer (10nM) – 1µL

CD-10 Reverse Primer (10nM) – 1µL

10x Buffer – 5µL

Q Solution – 10µL

dNTPs (25mM) – 2µL

Taq Polymerase – 0.5µL

Water – to 50µL

You have 2 samples plus a negative control that you need to test. How will you set up a master mix to make your pipetting more accurate and setting up this experiment go much faster than pipetting each individual component into 3 separate tubes? Remember to give yourself room for error.

d. While your PCR is running, you decide to go ahead and make your agarose gel. Your product size is expected to be 536bp. What percentage agarose gel do you need? How much agarose, and how much 1X TBE buffer to you need to accomplish this?

e. You pour your gel successfully, but use the last of the 1X TBE. You need more TBE to run the gel, but all we have is 50X TBE. You decide to make 3L of 1X TBE from the 50X TBE so that there is plenty for others (remembering your frustration from tissue culture and no PBS earlier in the day). How much 50X TBE do you need, and how much water do you need to make 3L?

3. Making Solutions:

a. You need 100mLs of a 5M solution of CaCl2. The MW of CaCl2 is 111 grams/mol. How many grams of CaCl2 do you need?

b. You want 25mLs of the following solution: 0.5M CaCl2 and 1M MgSO4. You have 5M CaCl2 and 2.5M MgSO4. How much of each do you add to make your final solution?

4. Many times, you will be using someone else’s lab notebook to formulate your own protocols. You will notice in some lab notebooks that the author will refer to nearly everything in volume rather than concentration. For example, it may say, “add 5μL DNA to the PCR tube” instead of “add 500ng DNA to the PCR tube.” Which is more accurate? Why?

After your frustration with tissue culture, you finally get your cells passaged and decide to set up your cDNA synthesis reaction, PCR, and agarose gel. You have extracted RNA from your cells, and now you need to proceed with the cDNA synthesis.

The first step is to determine the concentration of your RNA. You dilute your RNA 1:250, vortex it, move it to the cuvette, and run it on the spectrophotometer. The spec tells you that your concentration of RNA is 22µg/mL with an A260 of 0.32 and an A280 of 0.2. What is your ACTUAL RNA concentration, and what is its purity?

After determining your actual concentration of RNA, you are ready to make your cDNA. The protocol calls for the following for a total of 20µL:

5x iScript Reaction Mix – 4µL

iScript reverse transcriptase – 1µL

Nuclease free water – x µL

RNA template (100fg to 1ug total RNA) – x µL

You decide to use 500ng (or 0.5µg) of RNA in this reaction. How many µL of your RNA do you need, and how much water will you put in the tube?

You make the cDNA and get ready to set up your PCR for CD-10. The Qiagen protocol is as follows to a total of 50µL:

cDNA – 2µL

MgCl (25mM) – 4µL

CD-10 Forward Primer (10nM) – 1µL

CD-10 Reverse Primer (10nM) – 1µL

10x Buffer – 5µL

Q Solution – 10µL

dNTPs (25mM) – 2µL

Taq Polymerase – 0.5µL

Water – to 50µL

You have 2 samples plus a negative control that you need to test. How will you set up a master mix to make your pipetting more accurate and setting up this experiment go much faster than pipetting each individual component into 3 separate tubes? Remember to give yourself room for error.

While your PCR is running, you decide to go ahead and make your agarose gel. Your product size is expected to be 536bp. What percentage agarose gel do you need? How much agarose, and how much 1X TBE buffer to you need to accomplish this?

You pour your gel successfully, but use the last of the 1X TBE. You need more TBE to run the gel, but all we have is 50X TBE. You decide to make 3L of 1X TBE from the 50X TBE so that there is plenty for others (remembering your frustration from tissue culture and no PBS earlier in the day). How much 50X TBE do you need, and how much water do you need to make 3L?

Making Solutions:

You need 100mLs of a 5M solution of CaCl2. The MW of CaCl2 is 111 grams/mol. How many grams of CaCl2 do you need?

You want 25mLs of the following solution: 0.5M CaCl2 and 1M MgSO4. You have 5M CaCl2 and 2.5M MgSO4. How much of each do you add to make your final solution?

Many times, you will be using someone else’s lab notebook to formulate your own protocols. You will notice in some lab notebooks that the author will refer to nearly everything in volume rather than concentration. For example, it may say, “add 5μL DNA to the PCR tube” instead of “add 500ng DNA to the PCR tube.” Which is more accurate? Why?

Please answer questions at the end.

In this lab, you will add different volumes of “cell-free supernatant” (CFS) to fresh cultures of V. harveyi and observe bioluminescence. The CFS is prepared by culturing V. harveyi in liquid medium overnight, then separating the cells from the supernatant by centrifugation.

MATERIALS • overnight culture of V. harveyi in AB medium • fresh AB medium • 1 microcentrifuge tube • 3 glass culture tubes

PROCEDURE

1. This experiment will be performed with your lab group. Label the microcentrifuge tube and 3 glass cultures tubes with your group number. Also, label the 3 glass culture tubes as follows: “Media only,” “Cells+fresh media,” and “Cells+CFS.”

2. Using aseptic technique, pipet different volumes of fresh AB medium into the glass tubes as follows: 1 mL in the “Media only” tube, 800 ul in the “Cells+fresh media” tube, and 500 ul in the “Cells+CFS” tube.

3. Pipet 500 ul of the overnight culture into a microcentrifuge tube and centrifuge at maximum speed (>13,000 x g) for 5 min to pellet the cells.

4. After centrifugation is complete, transfer 300 ul of the cell free supernatant (CFS) from the microfuge tube to the glass tube labeled “Cells+CFS”. Be careful to avoid the cell pellet.

5. Remove the remaining supernatant from the microfuge tube using a micropipette and discard as culture waste.

6. Add 500 ul of fresh AB medium to the cell pellet in the microfuge tube and pipet up and down to resuspend until no clumps are visible.

7. Add 200 ul of the cell suspension to the “Cells+fresh media” tube and 200 ul of the cell suspension to the “Cells+CFS” tube. Note the time.

8. Place the 2 glass tubes containing cells in the 30 degree incubator/shaker.

9. After 1 hour and 2 hours, observe your tubes next to the “Media only” tube in a dark room. You will need to let your eyes adjust for at least 30 seconds (count to 30 slowly in the dark) before you can evaluate whether the cultures are luminescent or not. Also, make sure you try swirling the tubes in the darkroom and observe the tubes for a minute after you have stopped swirling. After the observation at the 1 hour time point, put your tubes back in the incubator shaker until the 2 hour time point.

10. Note your observations. At the end of the lab, place your tubes in the tube waste.

Questions

1. What were the expected results? Explain the rationale of the experiment. If you didn’t obtain the expected results, hypothesize why not and describe how the experiment could be improved.

2. What are differences between luminescence and fluorescence?

3. Why do V. harveyi cultures glow more brightly when swirled vigorously? Be specific.

4. Imagine you have a mutant V. harveyi strain that can’t synthesize autoinducer and another mutant V. harveyi strain that makes a defective luciferase. If you streaked both strains near (but not touching) each other on the same plate, would you expect to see light produced? If so, by which strain, where, and why?

5. You have two identical tubes of wild-type V. harveyi (with the same cell concentration in each tube) in fresh media. To one tube, you add CFS from a light-producing V. fischeri culture, and to the other tube you add an equal volume of fresh media. If both tubes of V. harveyi have the same light production, what can you conclude? Explain.

6. Many biomedical research labs study quorum sensing in V. fischeri or V. harveyi. How is studying light production in marine bacteria relevant to human health? List at least 3 different examples.

7. Why do you think it is important for bacteria to be able to control the expression of some genes based on the number of individuals present?

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