11/26/13

Today was the big day we ran our western blot and agrose gel. We first worked with our dilutions and then did the following steps:

Supplies and Reagents


SDS-Page Protocol

  1. Begin boiling water on hot plate.
  2. In a fresh, 1.5mL SCREW CAP tube add 15uL of your protein stock and 15uL of 2X Reducing Sample Buffer. Return your protein stock to the box in the -20C freezer labeled protein samples.
  3. Mix sample by flicking. Briefly centrifuge (10s) to pool liquid in bottom of tube.
  4. Boil sample for 5 mins.
  5. While sample is boiling, observe assembly of gel box and gels. Rinse gel wells thoroughly as demonstrated.
  6. When sample is finished boiling, immediately centrifuge for 1min. to pool liquid.
  7. Slowly load your entire sample into the appropriate well using a gel loading tip.
  8. Put lid on gel box and plug electrodes into appropriate receptacles on the power supply.
  9. Turn power supply on and set voltage to 150V. Run for 35mins. CHECK YOUR AGAROSE GEL RESULTS. MAKE SURE EVERYTHING IS SET UP FOR WESTERN BLOT.
  10. Turn off power supply and disconnect gel box from power supply.
  11. Remove lid from gel box.
  12. Disengage the tension wedge.
  13. Remove gel from gel box.
  14. Carefully crack open cassette to expose gel.
  15. Trim wells at top of gel.
  16. Notch a designated corner of the gel to help you remember the correct orientation of the gel (i.e. which is the top/bottom of the gel, which is the right/left side(s) of the gel)
  17. Proceed to Western Blotting protocol.

Western Blot:

Supplies and Reagents


Western Blot Protocol
  1. Soak the filter paper, membrane and gel in Tris-Glycine Transfer Buffer for 15 minutes.
  2. Assemble the blotting sandwich in the semi-dry blotting appartus:
    1. Anode (+++)
    2. filter paper
    3. membrane
    4. gel
    5. filter paper
    6. cathode (---)


  1. Transfer the blot for 30 minutes at 20V
  2. Remove the gel from the sandwich and rinse off adhering pieces of gel with transfer buffer.
  3. Put gel into blue solution and put on rotation plate
  4. Put membranes into 10 mL of blocking solution
  5. Let gels and membranes sit for 30 minutes
  6. Wash membrane 2 times, for 5 minutes each, with 20 mL of pure water.
  7. Wash gels with water.
  8. Put 10 mL of antibody on membranes and incubate overnight


11/25/13

Today I ran the quantification protocol for the protein samples. A note is that I had to run the quant process twice because the first time I put them in the spectrophotometer the machine was set to T as opposed A. The second time it was set correctly.

Protein Quantification Protocol
  1. Labeled 2 mL screw cap with names of samples on top and protein down the side.
  2. Dilute an aliquot of your protein sample 1:2 by pipetting 15uL of your protein sample into the 2 mL screw cap tube and the pipetting 15uL of DI water. Mix well by pipetting. Note: this dilution step is performed to ensure the sample absorbance falls within the range of the standard curve
  3. In a second 2 mL tube pipette 30uL of DI water (this tube will serve as your blank). Label tube as 'blank'
  4. To both tubes add 1.5mL of Bradford reagent.
  5. Invert the tubes several times and then incubate at room temperatire (RT) for 10mins.
  6. Mix the 'blank' via pippeting and transfer to a 1000ul to a plastic, disposable cuvette.
  7. Zero the spectrophotometer using your blank sample. Be sure to wipe the cuvette with a KimWipe first as any fingerprints or smudges can alter the reading.
  8. Mix the 'sample' via pippeting and transfer 1000 ul to a plastic, disposable cuvette
  9. Measure the absorbance at 595nm and record the value. Be sure to wipe the cuvette with a KimWipe first as any fingerprints or smudges can alter the reading.
  10. Remove the cuvette from the spectrophotometer. Using a P1000 set to 1000 ul, carefully pipette the solution in the cuvette up and down a couple of times to mix.
  11. Measure the absorbance at 595nm and record the value a second time.
  12. Average the two absorbance values you recorded.
  13. Back-calculate your protein concentration using the standard curve below. Hint: Use the equation on the graph provided. The relationship between absorbance and concentration is linear and defined by the equation y=mx+b. You have the average absorbance of your sample, x, and you want to calculate the concentration, y. Don't forget to account for the dilution in step 2!
  14. Protein samples went back into storage at -80 degrees C.
Name
 1
 2
 3
 Avg
U2 pre
 0.129
 0.129
 0.128
 0.128666667
C1 post
 0.034
 0.035
 0.036
 0.035
C1 pre
 0.191
 0.192
 0.192
 0.191666667
C2 pre
 0.079
 0.079
 0.079
 0.079
U1 post
 0.16
 0.163
 0.162
 0.161666667
U1 pre
 0.088
 0.088
 0.088
 0.088
U2 post
 0.089
 0.09
 0.09
 0.089666667
C2 post
 0.056
 0.057
 0.058
 0.057
Blank
 0
 0
 0
 0


11/22/13

Today was the first day of lab work. The only thing that I did was the Protein extraction part I. These samples were then put back into the freezer. Please see the following steps for the first part of extraction:

Supplies and Reagents

Protein Extraction Protocol
  1. Label the snap cap tube containing your tissue sample with your initials and the date using a lab marker.
  2. Add 500 ul of CellLytic MT solution to the 1.5mL snap cap tube containing your cut piece of frozen tissue.
  3. Homogenize the tissue with a sterile disposable pestle.
  4. Close the tube and invert the tube several times.
  5. Spin the tube in a refrigerated microfuge for 10 mins at max speed.
  6. While spinning, label a fresh tube with the word "Protein", source organism/tissue, your initials, and today's date.
  7. Carefully transfer supernatant (the clearish liquid on top) to labeled tube and store tube on ice.
After step seven the samples were put back into the -80 freezer for future use.

11/20/13

Today I was in charge of removing tissue samples from the urchins. I did not get as much from them as the first tissue sampling, but I was still able to get some. I just removed as many tube feet as I could as the urchins were in the bottom of their tanks. I labeled the tubes differently simply labeling them U1, U2, C1, C2 (U for Urchin, C for control). I also took pictures of the urchins so that I could match them with Jessica's so we made sure that they were able to match up from the first day. These samples went first on dry ice, and then to the -80 degrees C freezer in FTR in a box labeled "Team Heat Shock."

11/17/13

Today I was in charge of putting the species under the heat lamp, this means putting anemones for our other half of the group under the heat lamp. I put them in around 1:30 and the heat was around 22 degrees C. The process of removing the animals was simple just removing them from the water and letting the water drain from their tanks. They then went across the room and directly under the lamp. They sat there for 2 hours and then immediately put back into the water.

11/13/13

Today we began out experiment. First and foremost we cut our experiment way down. We went to four urchins total that me and Jessica will be working on. Two controls and two experimental. One experimental will go towards RNA extraction and one will go to protein synthesis. Jessica collected tube feet from the urchins before they were put under heat for the first treatment. Pictures were taken and sent to me so that I knew the labeling. That went as following for the urchins:
Control
 Experimental
U1 G
 U3 G
U1 P
 U3 P
U2 G
 U4 G
U2 P
 U4 P
11/5/13

The only thing we did today was collect urchins for Jessica. We got somewhere around 17 more urchins. They went into the same tanks with the rest of animals that were collected.

11/1/2013

Collection of animals at Southern Alki:

We arrived around 8:30 and were able to collect plenty of ochre stars (total of 16). Paul was able to collect quite a few sea anemones (around 26). Garrett amazingly was able to collect 5 nudibranchs. No urchins were able to be collected. All animals were gathered and put into buckets with water directly from the sound and then were transported to the tanks in the bottom of fisheries.

10/29/2013

Experimental set up for team Heat Shock Protein

- Four different organisms: Urchins, Sea Stars, Anemones, and Nudibranchs
- I will be working with Sea Stars

We will be putting the animals under a heat lamp once a day for two hours. We will do this for one week hopefully around the same time. We are still in the process of trying to figure out whether we want to put them in water under the lamp or have them exposed directly to the heat without any water over them.
We will be keeping all of our animals in the wet room in the basement of the Fisheries building with permission coming from Greg Jensen. We are going to take samples at the beginning, middle, and end of our experiment. Our goal is to have control and experimental so that we can have multiple animals for both PCR and protein analysis.

10/26/2013

Lab #4: Protein SDS/PAGE and Western blot, analyze conventional PCR, and qPCR data

Summary:
The purpose of this lab was to isolate proteins via western blot as well as look at lengths of DNA via electrophoresis. All measurements were taken using the oyster samples that were given to us in previous labs. PCR was run with the cDNA. An agrose gel was used in both instances. For the PCR and western blot electric currents were used to pull particles and seperate. In the end this lab was to ensure our techniques when our own experiment is performed.

Protein Extraction Part II Materials:


Protein Extraction Methods:

  1. Begin boiling water on hot plate.
  2. In a fresh, 1.5mL SCREW CAP tube add 15uL of your protein stock and 15uL of 2X Reducing Sample Buffer. Return your protein stock to the box in the -20C freezer labeled protein samples.
  3. Mix sample by flicking. Briefly centrifuge (10s) to pool liquid in bottom of tube.
  4. Boil sample for 5 mins.
  5. While sample is boiling, observe assembly of gel box and gels. Rinse gel wells thoroughly as demonstrated.
  6. When sample is finished boiling, immediately centrifuge for 1min. to pool liquid.
  7. Slowly load your entire sample into the appropriate well using a gel loading tip.
  8. Put lid on gel box and plug electrodes into appropriate receptacles on the power supply.
  9. Turn power supply on and set voltage to 150V. Run for 45mins. CHECK YOUR AGAROSE GEL RESULTS. MAKE SURE EVERYTHING IS SET UP FOR WESTERN BLOT.
  10. Turn off power supply and disconnect gel box from power supply.
  11. Remove lid from gel box.
  12. Disengage the tension wedge.
  13. Remove gel from gel box.
  14. Carefully crack open cassette to expose gel.
  15. Trim wells at top of gel.
  16. Notch a designated corner of the gel to help you remember the correct orientation of the gel (i.e. which is the top/bottom of the gel, which is the right/left side(s) of the gel)
  17. Proceed to Western Blotting protocol.

Western Blot Materials:


Western Blot Methods:
  1. Soak the filter paper, membrane and gel in Tris-Glycine Transfer Buffer for 15 minutes.
  2. Assemble the blotting sandwich in the semi-dry blotting appartus:
    1. Anode (+++)
    2. filter paper
    3. membrane
    4. gel
    5. filter paper
    6. cathode (---)


  1. Transfer the blot for 30 minutes at 20V
  2. Remove the gel from the sandwich and rinse off adhering pieces of gel with transfer buffer.
  3. Wash membrane 2 times, for 5 minutes each, with 20 mL of pure water.
  4. Put the membrane in the plastic box and add 10 mL of Blocking Solution. Cover and incubate overnight on a rotary shaker set at 1 revolution/second.
  5. Your TA will do the rest of the steps. After class tomorrow you can come and see your results.
  6. Decant liquid.
  7. Rinse the membrane with 20 mL of water for 5 minutes, then decant. Repeat.
  8. Incubate the membrane in 10 mL of Primary Antibody Solution. Decant the solution.
  9. Rinse the membrane with 20 mL of Antibody Wash for 5 minutes, then decant. Repeat 3 times.
  10. Incubate the membrane in 10 mL of Secondary Antibody Solution for 30 minutes. Decant.
  11. Wash the membrane for 5 minutes with 20 mL of Antibody wash, then decant. Repeat 3 times.
  12. Rinse the membrane with 20 mL of pure water for 2 minutes, then decant. Repeat twice.
  13. Incubate the membrane in 5 mL of Chromogenic Substrate until a purple band appears. This will occur between 1-60 minutes after adding the Chromogenic Substrate.
  14. Dry the membrane on a clean piece of filter paper to the open air.

Electrophoresis Methods:
  1. Place gel in gel box and fill with 1x TAE buffer (to fully cover wells)
  2. Remove combs from wells
  3. Load 7uL 100bp ladder in far left lane
  4. Load 20uL of your PCR sample into the gel (retain the remaining vol at -20ºC)
  5. Run gel at ~ 100V for ~ 1hr
  6. Visualize the gel on the UV transilluminator


10/19/2013

Lab #3: qPCR, analyze qPCR, Primer design, and prep for Experiment

Summary:

This weeks lab looked at qPCR and primer design. qPCR is used to quantify and target DNA molecules. We used our cDNA made last week for amplification. We quantified our data (but did not get these results back yet due to time constraints) using a computer, and our DNA was placed into an agrose gel. As stated before the purpose of this experiment was ultimately for practice but this practice is key to knowing how to amplify the DNA using the computer process, as well as how to set up and design primers for our experiments later on in lab. Note that this lab was not using the tradition PCR which quantifies the DNA at the end of the electrophoresis, but rather uses real time data for over an hour and a half to get results.

qPCR Materials:


qPCR Methods:

1. Prepare master mix: Prepare enough master mix for your number of reactions +1 to ensure sufficient volume recovery. (5 total reactions, 2 DNA samples, 2 Negative Controls)

For a 25μl reaction volume:
Component
 Volume
 Final Conc.
Master Mix (SsoFast EvaGreen supermix)
 12.5µL
 1x
upstream primer, 10μM
 0.5μl
 2.5μM
downstream primer, 10μM
 0.5μl
 2.5μM
Ultra Pure Water
 10.5uL
 NA

2. Add mastermix to wells of a white PCR plate
3. Thaw cDNA samples.
4. Add 1uL cDNA template to each reaction.
5. Add 1uL of ultra pure water to the negative control wells.
6. Cap the wells securely.
7. If necessary, spin the strips to collect volume in the bottom of the wells. (note: this was done upstairs before we loaded the tubes into the computer)
8. Ensure the lids are clean and place strips on ice. (I like to wipe the lids with a clean kimwipe)
9. Load the plate, verify the PCR conditions and start the run (this will be done by your TA).

PCR conditions:
1. 95°C for 10 minutes
2. 95°C for 15s
3. 55 °C for 15 s
4. 72°C for 15 s (+ plate read)
5. Return to step 2 39 more times
6. 95°C for 10s
7. Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

We did not see these results yet because we ran out of time. We will see them in next weeks lab.


Protein Extraction Materials:



Protein Extraction Methods:


Results:
From what I saw everything was successful. Because it was the end of class we did not get that great of looks at everything.

Summary:
Once again the purpose of this lab was to further our techniques for the lab we do in the future. I do not have any questions other than that I am looking forward to actually having results and getting to our experiment.

Results:

Because of time restraints we do not have any results. I briefly looked at the computer graph when the samples were still running, and my graph looked like it was supposed to. Other than that, we will not have any results until the next lab.


Summary:
The purpose of this lab was to prepare us for this type of quantification and protein extraction for our labs that we will conduct in the next few weeks. The computer measured the fluorescence in our cDNA samples. These tests would look at how pure our DNA was the sequence of our DNA for different things. I don't have any questions for this weeks lab because it was mainly running through steps and we were measuring much do to time constraints. Note we will be working with Anemones and nematocysts for our lab.


10/8/2013

Lab #2: RNA Isolation, Part 2

Summary:

Today’s lab was going through the process of isolating RNA from the tissue we started working with last lab. We used TriReagent in order to finish the isolation process. Once again I was using approximately 50-100 mg of Pacific Oyster gill. The purpose of this lab was for practice doing this RNA extraction technique so that it is perfected when our true lab experiments are conducted. TriReagent allows for separation of RNA from cell components and DNA, and it is a delicate process with many steps. This lab will further smooth the process, so that when our tests are conducted minimal errors will be made.

RNA Extraction Materials:

RNA Extraction Procedures:
These steps were continued from Lab 1:

1. Turn on heating block to 55°C.
2. Add remaining 500 uL of TriReagent to sample. (step was not done in the previous lab)
3. Incubate your homogenized tissue sample (from Lab 1) tube at room temperature (RT) for 5 mins.
4. In the fume hood, add 200uL of chloroform to your sample and close the tube.
5. Vortex vigorously for 30s. You are vortexing correctly if the solution becomes a milky emulsion.
6. Incubate tube at RT for 5 mins. (note: my tube incubated for much longer than 5 minutes because we were waiting for the microfuge in step 7 to become available.)
7. Spin tube in refrigerated microfuge for 15 mins. @ max speed.
8. Gently remove tube from microfuge. Be sure not to disturb the tube. (note: my tube was stuck in the microfuge... there may have been disturbance when trying to get it out)
9. Slowly and carefully transfer most of the aqueous phase (the top, clear portion) to a fresh microfuge tube. Do NOT transfer ANY of the interphase (the white, cell debris between the aqueous and organic phase).
10. Close the tube containing the organic and interphase and properly dispose of the liquid inside the tube as well as the tube itself at the end of the lab.
11. Add 500uL isopropanol to the new tube containing your RNA and close the tube.
12. Mix by inverting the tube numerous times until the solution appears uniform. Pay particular attention to the appearance of the solution along the edge of the tube. If mixed properly, it should no longer appear viscous/"lumpy". (note: my tube never looked viscous/lumpy, to the naked eye it looked uniform the entire time)
13. Incubate at RT for 10 mins.
14. Spin in refrigerated microfuge at max speed for 8 mins.
15. A small, white pellet (RNA and salts) should be present. If not, do not fret an continue with the procedure. (note: very very small pellet was present)
16. Remove supernatant.
17. Add 1mL of 75% EtOH to pellet. Close tube and vortex briefly to dislodge pellet from the side of the tube. If the pellet does not become dislodged, that is OK.
18. Spin in refrigerated microfuge at 7500g for 5mins. (note: once again this process was not done immediately after the previous step due to waiting)
19. Carefully remove supernatant. Pellet may be very loose. Make sure not to remove pellet!
20. Briefly spin tube (~15s) to pool residual EtOH. (note: this step was not done)
21. Using a small pipette tip (P10 or P20 tips), remove remaining EtOH.
22. Leave tube open and allow pellet to dry at RT for no more than 5mins.
23. Resuspend pellet in 100uL of 0.1%DEPC-H2O by pipetting up and down until pellet is dissolved.
24. Incubated tube at 55C for 5mins. to help solubilize RNA.
25. Remove tube from heat, flick a few times to mix and place sample on ice. This will be your stock RNA sample.
26. Quantitate RNA yield using Nanodrop spectrophotometer. (note: this step was not done because we moved on to cDNA procedures. TA said she trusted us)

cDNA Materials:


cDNA Methods

Results:

For the RNA extraction, I was successfully able to extract the DNA and get a write pellet at the bottom of my tube. Because I did not quantify my sample, the accuracy of the sample is unknown, but based off of my DNA extraction I bet they are pretty good. cDNA is unknown at this time because we have not yet gotten our samples back yet.

Summary:

The purpose of this lab was to further our skills in this area in order to have efficient and accurate data for our experiments later in lab. This will be key for our later experiment, so to get the steps and practice them will be a big help. We did not really measure anything in this lab, but the steps taken will be a step in order to get cDNA which we will be able to get results for. RNA extraction is essential for cDNA production as well as isolation steps to get rid of other cell components as well as DNA. Nothing was really unclear throughout the extraction. It would be nice to know how longer incubation periods affect the data, because there were often times when we stood for much longer then was said. If taking longer between steps affects the final product, then there will need to be times when I as well as my fellow classmate work faster in order to ensure we get the most accurate RNA and DNA.

Possible Lab experiment:
This is just a note to myself. I want to look at DNA differences in Cnidarian nematocysts (in particular Jellyfish), from the same genus but different geographic locations (north sound vs south sound). If these differ, it may lead to knowledge of feeding as well as interactions with predators. I am reading about minicollegians and different proteins that work to fire the nematocysts and this may lead to steps used above in order to figure them all out.

10/6/2013

Lab #1: DNA isolation; initiate RNA isolation


Summary:

For this lab we used oyster tissue samples in order to begin the RNA extraction process. As well as extracting RNA, we used the oyster tissue in order to isolate and quantify DNA from these tissue samples. In terms of the RNA extraction Tri-Reagent was used in order to separate the RNA from other components of the cell. Only the first steps of this process were carried out in the lab due to time constraints, and the samples were frozen in order to proceed with the rest in proceeding labs. For the DNA sample a DNazol extraction kid was used in order to precipitate the DNA from the cell. All steps were carried out in this lab period for DNA extraction and ultimately DNA quantification protocol was used at the end in order to see accuracy of the DNA extracted. As stated before, oyster tissue was used to carry out both RNA and DNA extraction. For the DNA extraction Olympia Oyster Gill was used, and for RNA Pacific Oyster gill was used. Approximately 25-50 mg of tissue was used for the DNA extraction, and 50-100 mg of the tissue was used for the RNA extraction.

RNA Extraction Materials:

RNA Extraction Methods:


Three proceeding steps were not completed this lab, but will be finished for the during the next lab period. After the tissue sample was homogenized it was put in the freezer in order to preserve it for the next lab
DNA Extraction Materials:

DNA Extraction Methods:
DNA Quantification Materials

DNA Quantification Methods

Steps above were preformed once by the first student to use the nanodrop. Because we are all using the same DNA solution it I was not necessary to preform those particular step for each person.
Results:

Sample #: 8
Abs:10.401
A-260: 4.774
A-280: 2.567
260/280: 1.86
260/230: 0.46
ʎ: 230

Conclusion:

Based off of what the TA told me, my results were good and were close to expectations The 260/280 is suppose to be as close to 2.0 as possible, and because mine was at 1.86 that is reasonably close. My DNA had little ethanol which means that my cleaning process went well. Some errors that may have occurred would have been in the incubation period during which I was waiting for a centrifuge to open. For this reason the DNA sat for longer than expected. As well during the beginning stages, my sample was not on ice the entire time, and the top was left open, leading to a possibility of contaminants entering the test tube. The RNA portion of the experiment has no results nor can the results be determined until the experiment is completed. Possibilities for next steps would be PCR in order to amplify and copy the DNA for testing.
Reflection:
To me the purpose of this lab was to isolate both RNA and DNA from other components of the cell. More importantly the purpose of this lab is to learn the techniques of extraction in order to be confident if we need to use this for our experiments later in the quarter. The procedures in this lab are not necessarily there to measure anything but rather for extraction purposes. The quantification of the DNA is there to measure the amount of DNA extracted during the process. This is used in order to take DNA and RNA from cells in order to then create copies and run tests on it. This is useful for genome projects as well as to study all sorts of diseases. Because DNA is the building block of life, there are quite a few things that are studied from it. Extracting the DNA is the first step in order to study it. This lab was pretty straight forward, so I do not have any burning questions about it. I do wonder what the maximum amount of DNA you can extract at a time is? You obviously cannot extract all DNA and RNA from the human body and there must be a max of cells you can perform on at a certain time, so what is it? For forensics, not necessarily in the marine environment, how small can it be as well? Other than that the lab was straight forward and informative.

Genes that I want to study:

The hox gene. I want to look at this because I know that this gene plays an important role in segmentation of embryos and limb development in both humans and fruit flies, but I have no idea how the hox gene works in marine organisms. For this reason I want to look more into it.

Amyloid Precursor Protein. Although far fetched, this is the gene product for alzheimer's disease. It would be interesting to see if other marine species even have the capabilities to have diseases of this sort. Although we aren't studying marine mammals, but dolphins come to mind. You would think that if you could find this gene product in something as small as an anemone or urchin it could be groundbreaking.

Growth gene for calcium carbonate shells - It would be cool to isolate a gene for the growth of shells in order to counter Ocean Acidification. With the oceans heading the way they are it would be interesting to find a gene that would be able to counter the affects that are happening right now. If you could isolate the gene so that the shellfish could produce some sort of alternative shell as oppose to one that would degrade over time you could fix all of the shellfish problems that are happening due to ocean acidification.