Talk:20.109(S13):Module 3

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===Part 2: Microscopy===
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==Protocols==
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When observing your cells under fluorescence excitation, you should work with the room lights off for best results. A member of the teaching faculty will be with you to help you make the most of your 20-25 minutes.
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<font color=purple>If you got to go to the TC room first on Day 2, you will go in the second cohort today (and vice-versa). If you are in the second group, use the time that you are waiting to complete your research idea discussion, and if you have time to also prepare your RNase-free area, label tubes that you will need, etc.</font color>
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#Prior to the first group using the microscope, the teaching faculty will turn on the microscope and allow it to warm up for 15-20 min. First, on the mercury lamp that is next to the microscope, the ‘POWER’ switch will be flipped. Next, the ‘Ignition’ button will be held down for about a second, then released.  
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===Part 1: Research idea discussion===
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#When you arrive, the ''lamp ready'' and ''power'' indicators should both be lit – talk to the teaching faculty if this is not the case.  
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#Place your first sample slide on the microscope, coverslip-side up, by pulling away the left side of the metal sample holder for a moment. 
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Before (second TC cohort) or after (first TC cohort) your wet-lab work today, take some time to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below.  
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#Begin your observations with the 10X objective.  
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[[Image:20109_fluor-scope_front.jpg|thumb|left|250px|Fluorescent microscope, front view.]]
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Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you’ve gathered. Consider them based on:
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[[Image:20109_fluor-scope_side.jpg|thumb|center|250px|Fluorescent microscope, side view.]]
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* your interest in the topic
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* the availability of good background information
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#Turn on the illumination using the button at the bottom left of the microscope body (on the right-hand side is a light intensity slider).  
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* your likelihood of successfully advancing current understanding
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#Next, turn the excitation light slider at the top of the microscope to ‘DIA-ILL’ (position 4).
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* the possibility of advancing foundational technologies or finding practical applications
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#Try to focus your sample. However, be aware that the contrast is not great for your cells, and you might not be able to focus unless you find a piece of debris. Whether or not you find focus, after a minute or two, switch over to fluorescence. Your cells will be easier to find this way.
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* if your proposal could be carried out in a reasonable amount of time and with non-infinite resources
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#*First, turn the white light illumination off.
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#*Next, move the excitation slider to ‘FITC’ (position 3). You should see a blue light coming from the bottom part of the microscope.  
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It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but by the end of today’s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic. '''Check in with Thomas and get his feedback about your ideas for a few minutes before leaving today.'''  
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#**This light can excite both the green and the red dye in the viability kit, and the associated filter allows you to view both colors at once.  
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#*Finally, you must slide the light shield (labeled ‘SHUTTER’) to the right to unblock it. Now you can look in the microscope, and use the coarse focus to find your cells (which should primarily be bright green), then the fine focus to get a clearer view.
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A few ground rules that are 20.109 specific:
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#*You can also switch the excitation slider over to ‘EthD-1’ (position 2) to see only the red-stained cells. Some of your cells may appear to be dimly red, but the dead ones are usually obviously/brightly stained.
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*You should not propose any research question that has been the subject of your UROP or research experience outside of 20.109.  This proposal must be original. 
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#*'''Be aware that the dyes do fade upon prolonged exposure to the excitation light, so don’t stay in one place too long, and when you are not actively looking in the microscope, slide the light shield back into place.'''
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*You should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a twelve minute oral presentation.
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#You can try looking at your cells with the 40X objective as well if you have time. As you move between objectives and samples, choose a few representative fields to take pictures of. '''As a minimal data set, try to get 3 fields at 10X of both of your samples.'''
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#*To take a picture, remove one eyepiece from the microscope, and replace it with the camera adaptor. Be sure to keep the light shield in place until you are ready to take the picture (to avoid photobleaching)!
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Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the [[Yeast rebuild |“yeast rebuild”]] or the [[T7.2 | “T7.2”]] wiki pages on OpenWetWare for examples of research ideas in process. As part of a later FNT assignment, you will have to print out your wiki page specifying your topic, your research goal and at least two helpful references that you’ve read and summarized.
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#*Note that 10X images will reveal a broader field, but 40X images may have better contrast.  
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#*Check with the teaching faculty if you are having difficulty getting clear pictures.
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===Part 2: Prepare cell lysates===
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#*Later in the module, you will compare the average cell numbers in each sample using the statistical methods we discussed during Module 2.
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#Post two well-captioned pictures to the wiki before leaving (one of each sample), so we can discuss the class data in our next lecture. Be sure to note whether the image is at the surface or core of the bead.
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You will prepare cell-bead samples in three different ways: one will allow  you to count your cells, and is suitable for RNA preparation, while the other two will involve more stringent bead/matrix dissolution for better protein or proteoglycan recovery. Split up the work with your partner whatever way is most convenient. '''Remember to label your samples carefully at every step.'''
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#*If you are one of the last two groups to use the microscope, you may post your data within 24 hours instead.
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 +
#Before proceeding, briefly observe the cell-bead constructs under the microscope and note any changes from Day 3.
 +
#*Let the teaching faculty know if you have difficulty focusing within a bead.  
 +
#Remove the culture medium from each of your samples. Be careful not to suck up the beads; it will help to use a serological pipet just as you did when washing your freshly synthesized beads. Tipping the plate will help the beads settle in a cluster and allow you to remove medium elsewhere.
 +
#*A 5 mL pipet size should work well for rigid beads, while for more delicate beads, you should use a 2 mL serological pipet or even a P1000. If your beads are falling apart, you can transfer the beads according to steps 3-5 below without trying to remove medium first.
 +
#*If you are concerned about your bead amount, talk to the teaching faculty. You might skip the proteoglycan assay and focus on the other two instead.
 +
#About 1/3 of your beads will be used to measure protein content: move these to an eppendorf tube. The goal is about 10-15 (2-3 mm) beads per tube.
 +
#*For large beads (4-5 mm), you might use only 5-10 beads, and for very small beads (<1 mm), you might use 20 or more.  
 +
#Another 1/3 will be used to measure proteoglycan content; these whole beads can also be moved to an eppendorf tube.
 +
#The final 1/3 will be used to isolate RNA. Using a sterile spatula, transfer the beads into a fresh well of your 6-well plate. This transfer step is to exclude any cells that are growing on the bottom of the plate (as opposed to actually in the beads) from analysis.
 +
 
 +
====Samples for RNA Isolation====
 +
 
 +
#Rinse the transferred bead-cell constructs with 4 mL of warm PBS, then aspirate the buffer.
 +
#*If your beads are very fragile, you might want to skip the PBS rinse, and directly proceed to step 2.
 +
#Add 3 mL of pre-warmed EDTA-citrate buffer, and incubate at 37 &deg;C for 10 min.
 +
#*Meanwhile, prepare the beads for the protein and proteoglycan assays as described below. All the materials that you need are in eppendorf tubes in the fridge.
 +
#Now recover your cells:
 +
#*Add 3 mL of warm complete culture medium, pipet up and down to break up the beads (you may find this easier with a 1 mL pipetman rather than a serological pipet), and transfer to a 15 mL conical tube.  
 +
#*Spin the cells down at 1900g for 6 min (using the centrifuge that is in the TC room).
 +
#Resuspend in ~ 1-1.5 mL of culture medium, and ''write down'' what you use. Mix thoroughly by pipetting, then set aside a 90 &mu;L aliquot of your cells for counting, and put the rest of the cells into another eppendorf tube.
 +
#*If you have very few cells based on your Day 3 observations and/or having very few beads, you might consider skipping the cell count, and instead keeping all of the cells for RNA isolation. If you have too few cells to get a reliable cell count, you are not losing valuable information for your report in any case. And if you have so few cells that taking some of them for a count compromises your other data, then that outcome would not be preferable to missing the cell count.  
 +
#While one of you begins the spin in the main lab (see Part 2), the other should count your cell aliquot as on Day 2, at a 9:1 ratio with Trypan blue. '''Separately''' calculate the approximate numbers of live (yellowish) and of dead (blue) cells.
 +
#*Recall that you must multiply by 10,000 (and your dilution factor) to convert a hemacytometer cell count to a cells/mL concentration.
 +
 
 +
====Samples for Protein Extraction====
 +
 
 +
#Per eppendorf tube (typically 10-15 beads), add 133 &mu;L of cold EDTA-citrate buffer, and pipet up and down for 20-30 seconds to dissolve the beads. Be thorough while limiting bubbles as best you can. The resulting solution may be viscous.
 +
#Pipet 33 &mu;L of 0.25 M acetic acid into each eppendorf tube.
 +
#Finally, pipet 33 &mu;L of 1 mg/mL pepsin (in 50 mM acetic acid) into each tube and mix well.
 +
#Move your eppendorf tubes into the rack in the 4 &deg;C fridge. Tomorrow the teaching faculty will move them to an elastase solution (also at 4 &deg;C) to break down the polymeric collagen to more readily measured monomeric collagen.
 +
 
 +
====Samples for Proteoglycan Extraction====
 +
 
 +
#Soak your beads for a few minutes in pre-warmed PBS, and then remove as much of the PBS as possible. Shoot to have '''no''' pink tint to the beads, as it is known to interfere with the proteoglycan assay.
 +
#Add 250 &mu;L of papain solution to your beads. The papain is in an EDTA-citrate buffer base.
 +
#When the first partner goes to the main lab, s/he should take this tube to the 60 &deg;C heat block. After 24 hours, the samples will be moved to the fridge.

Current revision

Contents

Protocols

If you got to go to the TC room first on Day 2, you will go in the second cohort today (and vice-versa). If you are in the second group, use the time that you are waiting to complete your research idea discussion, and if you have time to also prepare your RNase-free area, label tubes that you will need, etc.

Part 1: Research idea discussion

Before (second TC cohort) or after (first TC cohort) your wet-lab work today, take some time to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below.

Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you’ve gathered. Consider them based on:

  • your interest in the topic
  • the availability of good background information
  • your likelihood of successfully advancing current understanding
  • the possibility of advancing foundational technologies or finding practical applications
  • if your proposal could be carried out in a reasonable amount of time and with non-infinite resources

It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but by the end of today’s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic. Check in with Thomas and get his feedback about your ideas for a few minutes before leaving today.

A few ground rules that are 20.109 specific:

  • You should not propose any research question that has been the subject of your UROP or research experience outside of 20.109. This proposal must be original.
  • You should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a twelve minute oral presentation.

Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the “yeast rebuild” or the “T7.2” wiki pages on OpenWetWare for examples of research ideas in process. As part of a later FNT assignment, you will have to print out your wiki page specifying your topic, your research goal and at least two helpful references that you’ve read and summarized.

Part 2: Prepare cell lysates

You will prepare cell-bead samples in three different ways: one will allow you to count your cells, and is suitable for RNA preparation, while the other two will involve more stringent bead/matrix dissolution for better protein or proteoglycan recovery. Split up the work with your partner whatever way is most convenient. Remember to label your samples carefully at every step.

  1. Before proceeding, briefly observe the cell-bead constructs under the microscope and note any changes from Day 3.
    • Let the teaching faculty know if you have difficulty focusing within a bead.
  2. Remove the culture medium from each of your samples. Be careful not to suck up the beads; it will help to use a serological pipet just as you did when washing your freshly synthesized beads. Tipping the plate will help the beads settle in a cluster and allow you to remove medium elsewhere.
    • A 5 mL pipet size should work well for rigid beads, while for more delicate beads, you should use a 2 mL serological pipet or even a P1000. If your beads are falling apart, you can transfer the beads according to steps 3-5 below without trying to remove medium first.
    • If you are concerned about your bead amount, talk to the teaching faculty. You might skip the proteoglycan assay and focus on the other two instead.
  3. About 1/3 of your beads will be used to measure protein content: move these to an eppendorf tube. The goal is about 10-15 (2-3 mm) beads per tube.
    • For large beads (4-5 mm), you might use only 5-10 beads, and for very small beads (<1 mm), you might use 20 or more.
  4. Another 1/3 will be used to measure proteoglycan content; these whole beads can also be moved to an eppendorf tube.
  5. The final 1/3 will be used to isolate RNA. Using a sterile spatula, transfer the beads into a fresh well of your 6-well plate. This transfer step is to exclude any cells that are growing on the bottom of the plate (as opposed to actually in the beads) from analysis.

Samples for RNA Isolation

  1. Rinse the transferred bead-cell constructs with 4 mL of warm PBS, then aspirate the buffer.
    • If your beads are very fragile, you might want to skip the PBS rinse, and directly proceed to step 2.
  2. Add 3 mL of pre-warmed EDTA-citrate buffer, and incubate at 37 °C for 10 min.
    • Meanwhile, prepare the beads for the protein and proteoglycan assays as described below. All the materials that you need are in eppendorf tubes in the fridge.
  3. Now recover your cells:
    • Add 3 mL of warm complete culture medium, pipet up and down to break up the beads (you may find this easier with a 1 mL pipetman rather than a serological pipet), and transfer to a 15 mL conical tube.
    • Spin the cells down at 1900g for 6 min (using the centrifuge that is in the TC room).
  4. Resuspend in ~ 1-1.5 mL of culture medium, and write down what you use. Mix thoroughly by pipetting, then set aside a 90 μL aliquot of your cells for counting, and put the rest of the cells into another eppendorf tube.
    • If you have very few cells based on your Day 3 observations and/or having very few beads, you might consider skipping the cell count, and instead keeping all of the cells for RNA isolation. If you have too few cells to get a reliable cell count, you are not losing valuable information for your report in any case. And if you have so few cells that taking some of them for a count compromises your other data, then that outcome would not be preferable to missing the cell count.
  5. While one of you begins the spin in the main lab (see Part 2), the other should count your cell aliquot as on Day 2, at a 9:1 ratio with Trypan blue. Separately calculate the approximate numbers of live (yellowish) and of dead (blue) cells.
    • Recall that you must multiply by 10,000 (and your dilution factor) to convert a hemacytometer cell count to a cells/mL concentration.

Samples for Protein Extraction

  1. Per eppendorf tube (typically 10-15 beads), add 133 μL of cold EDTA-citrate buffer, and pipet up and down for 20-30 seconds to dissolve the beads. Be thorough while limiting bubbles as best you can. The resulting solution may be viscous.
  2. Pipet 33 μL of 0.25 M acetic acid into each eppendorf tube.
  3. Finally, pipet 33 μL of 1 mg/mL pepsin (in 50 mM acetic acid) into each tube and mix well.
  4. Move your eppendorf tubes into the rack in the 4 °C fridge. Tomorrow the teaching faculty will move them to an elastase solution (also at 4 °C) to break down the polymeric collagen to more readily measured monomeric collagen.

Samples for Proteoglycan Extraction

  1. Soak your beads for a few minutes in pre-warmed PBS, and then remove as much of the PBS as possible. Shoot to have no pink tint to the beads, as it is known to interfere with the proteoglycan assay.
  2. Add 250 μL of papain solution to your beads. The papain is in an EDTA-citrate buffer base.
  3. When the first partner goes to the main lab, s/he should take this tube to the 60 °C heat block. After 24 hours, the samples will be moved to the fridge.
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