Summery: There is no ordinary lab, we were just answering questions about our experiments.
1. Detail at least 2 reasons why your results turned out the way they did. This should be easy to do if your results are "unexpected", but even expected results can have multiple explanations. Really think about this, the answer "because I messed up in lab" (or any variation thereof) is not acceptable. My results for western blot had two bands . These bands were hardly visible but they were present and it would be reasonable to conclude that Hsp70 protein expression was present in gonad tissue and in protein tissue samples. The reason why the bands weren't purple enough could indicate some sort of contamination or due to the fact that gel was left running for longer than it was supposed to the bands turn out to be much lighter than i anticipated.
I can't think of
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2. What are two obstacles that you encountered during your lab work and experimental design? Did these obstacles affect your results? Why? The number one obstacle that I can think of is making little errors on pipetting, especially when making significantly small transfers. What I noticed was that sometimes there was still some liqued left in the tube or by pressing on the pipette harder more liqued was transfered. Second obstacle was lacking organizing skills such as taking notes and recording all the observations while performing any experiments. I felt that was very important part of the lab but sometimes I failed to do so and it certainly effected my results because I relied on my memory.
3. Explain at least one aspect of your research and its results that have a greater impact outside of your own personal learning experience. What would you tell a non-scientist who challenged the importance of your research? My results show that sea urchins that were exposed to higher amount of stress had a greater hsp 70 protein expression and this was exactly what I have anticipated. Stressing out the importance of the research to a person who is non-scientist will better inform them about the great experience behind the research and its importance predicting and making sientific theories.
4. What part of your research and analysis has completely stumped you? Is there anything you can do to find the answer or will it always remain a mystery? I just wonder why the bands turned out so bright. I did two experiments on western blot and had same (bright bands) each time.
5. In about 3 sentences each, summarize 2 papers that you are going to cite in your own paper that give insight into the results that you found. One paper talks about few important concepts, one is how the hsp70 are translated from the RNA. I believe that this should prvide me with some great information that will help me with my discussion for the research paper. Another paper talks about the importance and benefits of studying more about sea urchins.
Date: November 22, 2011
Summary: The purpose of this lab was to do another Western Blot to get a more accurate results concerning HSP70 expression. This is the second Western Blot that I have performed for my research. the whole process took me two days; Tuesday and Thursday.
Materials and Methods:
I used same materials and methods as I did previously for Western Blot
Results:
- Dwell 7 (S x G) HSP70 present
- Dwell 8 (C x G) HSP70 no expression
- Dwell 9 (P x G) HSP70 present
- Dwell 10 (P x D) HSP70 no expression
- Dwell 11 (C x D) HSP70 no expression
- Dwell 12 (S x U) HSP70 no expression
The bands on the membrane were barely visible on dwell 7 and 9 but they were still there. S stands for solinity and predator and G stands for gonad, P stands for predator...
I had the same results for my first Western Blot test.
Conclusion: This lab went pretty smooth since I used same methods and materials. However I had to prepare master mix by myself which was very interesting. This experiment actually took two days to complete but it was all worth it and I've got sufficient results now.
Reflection: I think this was the last experiment that I will have to do for my research since my results were the same from my first western blot test and the second.
Date: November 15, 2011
Summary: During this lab we worked on techniques that should help us with our research. Specifically we used DNA samples to do a Dot Blot, and cDNA samples to do Q PCR.
Material and Methods:
Dot Blot
- Use calculation on board: V1=50x40/168.8 V1=17.108 so I was using this amount in uL of DNA sample and 23uL of water. Total was 40 uL
- 0.8 ng/ul = 124ul H2O, 60ul of 20X SSC, 16ul of DNA (50ng/ul)
- 0.4 ng/ul= 132ul H2O, 60ul of 20X SSC, 8ul of DNA (50ng/ul)
- 0.2 ng/ul= 136ul H2O, 60ul of 20X SSC, 4ul of DNA (50ng/ul)
- 0.1 ng/ul= 138ul H2O, 60ul of 20X SSC, 2ul of DNA (50ng/ul)
- 0.05 ng/ul= 139ul H2O, 60ul of 20X SSC, 1ul of DNA (50ng/ul)
- Cut out nylon membrane to fit a 72 well manifold
- Soak membrane in 6x ssc buffer for 10 minutes
- Cut filter paper so its size is equivalent to membrane and soak it in 6x ssc buffer
- Paper filter goes on the bottom and membrane on top
- Use screw cap tube to denature DNA sample for 10 minutes in boiling water and after that transfer sample to ice
- Apply 500ul of 6X SSC buffer to all the wells in the manifold and switch on vacuum to allow SSC to filter through
- Centrifuge the sample for 5 minutes
- When putting each DNA sample into wells, don not touch the membrane, and know which wells are yours
- Allow the sample to filter through membrane, use pipette when it is not filtering
- Transfer membrane dot side up to filter paper
- For 10 minutes, soak on denaturation buffer and then n neutralization buffer for 5 minutes
- Always place the membrane on dry filter paper to dry
- Wrap dot blot in plastic and place DNA side down on the UV for 2 minutes at 120 kj
Washing:
- Prepare blocking solution and antibody solution
- Place membrane in 10 ml blocking solution for about 30 minutes incubation and place on rotary shaker
- Empty blocking solution and rinse membrane with 20ml of water for 5 minutes
- Incubate membrane with 10ml of primary antibody for 1 hour
- Empty antibody solution and wash membrane with 20mol of 1x TBS-T for 5 minutes (repeat 3 times)
- Incubate with secondary anibody solution for 30 minutes and decant
- Wash membrane for 5 minutes with 20ml of TBS-T, repeat 3 times
- Rinse with 20ml of H2O for about 2 minutes and repeat this 2 times
- Incubate with 5 ml of Chromogenic Substrate until color develops
- Rinse membrane with 20ml of H2O for about 2 minutes and emty it, repeat it 2 times
- Use filter paper to dry the membrane on it
QPCR:
- Obtain a cDNA sample
- Make master mix: 12.5x5=62.5; 1x5=5; 1.25x5=6.25; 1.25x5=6.25; 7x5=35 (2x immomix, syto, forward primer, reverse primes, water respectively)
- Add 23ul master mis to wells of white pcr plate
- Add 2ul of water to each negative control
- Cap wells securely
- Spin down the liquid pools at the bottom
- Load the plate and verify the PCR conditions, then start the run
PCR conditions:
- 95C for 10 minutes
- 95C for 15 sec
- 55C for 15 sec
- 72C for 30 sec
- Return to step 2 about 39 more times
- 95 C for 10 sec
- Melt curve from 65C to 95 C at 0.5C sec
Results:DNA methylation dot blot seems to have positive results. We will discuss these results in greater details in the next lab
Conclusion: Didn’t really have time to finish this experiment but went well overall
Reflection: This lab was aimed to test for DNA methylation, it was a very hectic lab and I hope next lab will go much better for me.
Date: November 8, 2011
Summary: This was the first lab where we began working independently on our group project. I’ve started working on the protein extraction to ultimately measure the expression of HSP 70 by using SDS Page/Western Plot. At first I started off with RNA Extraction, the procedure and methonds can be found in my lab notebook in the first lab.
SDS Gel:
- Add 15uL of protein that was extracted and 15uL of 2x reducing sample buffer
- Mix it well and centrifuge to pool liquid
- Boil the sample for 5 minutes
- After boiling, centrifuge it for 1 minute
- Add 7 uL of latter to one end of the gel
- Add 30 uL of protein extract sample to each well of the gel and ran gel for 25 minutes at 150 V
Western Blot
- When assembling the blotting sandwich, use a filter paper; membrane; gel; more filter paper. Place the sandwich on top of positive anode and thoroughly soak in trisglycine transfer buffer
- Place the cathode on top and run the blow for about 30 minutes at 20 V
- Remove the gel from the membrane and place it in a staining solution inorder to make the protein visible
- Rinse the membrane about two times in super pure water by placing in the tray of water on a shaker plate for about 5 minutes
- Place the membrane in a new tray and cover with blocking solution
- Incubate the tray overnight on a shaker plate set to 1 revolution per minute
- Rinse the membrane with 20 ml of water for 5 minutes twice
- Wash the membrane with primary antibody solution (HSp-70) three times for five minutes at a time
- Incubate membrane with 10ml of secondary antibody solution for 30 minutes, two times
- Also, incubate in chromogenic solution until purple bands become visible
- Dry the membrane in open air
- Observe the membrane and determine what proteins are present
Important Notes
- After denaturation of my proteins was complete, I and two other students assembled the box and prepared the gel and added 1xTAE buffer submerging the gell and the electrodes.
- Originally I had 6 denatured protein samples but I ended up pipetting only 3 of them into wells 10-12 on SDS Page.
- We ran 150V through the gel for 45 minutes, but I’ve left the lab after 5 minutes when the current started running through the gel so I don’t really know yet what happened next.
Results: As I mentioned I haven’t seen how my samples looked in the gel yet.
Conclusion: The first thing I have to do next time is look at the gel and see how my samples look and make some observations. Other than that I was able to finish what I planned out to do during that experiment.
Reflection: At first I was little clueless on what to do, but with some help I was able to figure out what procedures I needed to do and I’ve done everything successfully. This research is getting very interesting but also intense.
Date: November 1, 2011
Summery: In this experiment we did a preparation of urchin gonadal tissue for protein extraction. We also ran a cDNA samples IGF1 and IGF2 on gel for 45 minutes to determine presence and length of the product. During the last aspect of the lab, we went over western blot procedures where a protein extraction was used, processed and placed on a gel.
Methods and Matherials:
Protein Extraction:
- Get a 1.5ml snap cap tube and label it (IS, 11,01,11. Protein)
- Obtain 0.030g of Gonad sample and place it in the fresh tube that was just labeled
- Add 500 ul of CellLytic MT to the tube
- Use a sterile pestle to homogenize the mixture and shake /invert it few times.
- Spin the tube in a centrifuge for ten minutes at a maximum speed
- Label another fresh 1.5 snap cap tube with “Protein” and IS and date
- After the centrifuge, pipett the supernatant out of the original tube and place it into a new tube
- Put a 1.5% agrose gel in gel box and cover it with 1xTAE buffer
- Load 7uL of ladder to wells on both ends of both rows
- Load 25uL of PCR product into each well after which ran the gel for 60 minutes at 100 V
- Observe the gel in UV box to determine length and what was aplfilied
SDS Gel:
- Add 15uL of protein that was extracted and 15uL of 2x reducing sample buffer
- Mix it well and centrifuge to pool liquid
- Boil the sample for 5 minutes
- After boiling, centrifuge it for 1 minute
- Add 7 uL of latter to one end of the gel
- Add 30 uL of protein extract sample to each well of the gel and ran gel for 25 minutes at 150 V
Western Blot
- When assembling the blotting sandwich, use a filter paper; membrane; gel; more filter paper. Place the sandwich on top of positive anode and thoroughly soak in trisglycine transfer buffer
- Place the cathode on top and run the blow for about 30 minutes at 20 V
- Remove the gel from the membrane and place it in a staining solution inorder to make the protein visible
- Rinse the membrane about two times in super pure water by placing in the tray of water on a shaker plate for about 5 minutes
- Place the membrane in a new tray and cover with blocking solution
- Incubate the tray overnight on a shaker plate set to 1 revolution per minute
- Rinse the membrane with 20 ml of water for 5 minutes twice
- Wash the membrane with primary antibody solution (HSp-70) three times for five minutes at a time
- Incubate membrane with 10ml of secondary antibody solution for 30 minutes, two times
- Also, incubate in chromogenic solution until purple bands become visible
- Dry the membrane in open air
- Observe the membrane and determine what proteins are present
Results: The Gel Electrophoresis produced 3 visible bands which were all around 200bp. The bands were found in two samples and one control well.
Conclusion: The results from gel show amplification around 200 range so it is consistent with the size of the product that was used for the primers: Gigasin-2 F and R for HSP70. In our controls, we noticed that one of them was contaminated.
Reflection: This lab was by far the most interesting one, especially the Gel Electrophoresis demonstration. I just wish we could have more time to better understand the steps of this technique. Other than that it was a great lab and all what we learn should be a useful tool in our personal research.
Date: October 25, 2011
Summary: Having the cDNA which was prepared from the previous lab experiment and primers that were ordered, do the PCR on cDNA by using Thermocycler. PCR technique is used to replicate a gene of interest for further studying. We also dissected urchin: one from each group and collected appropriate amount of tissue.
Materials:
- Micropipettes (20, 200, 1000 microliters)
- TE Buffer
- Gloves
- Goggles
- Waterbath
- 0.5mL PCR tubes
- 1.5mL microcentrifuge tubes (RNAse free)
- dTNPs
- cDNA sample (C. Gigas)
- 5x Go Tag Green buffer
- Primers
- Nuclease Free water
- Microcentrifuge
- Thermocentrifuge
Methods:
- When preparing primers, centrifuge for about 3 minutes at 7500 rpm
- Add 269 microliters of TE buffer
- Warm it up for 1 minute in 45 C water bath
- Vortex briefly for about 5 seconds
- Centrifuge for about 3 minutes at 7500 rpm
- Vortex again
- Label 1.5mL microcentrifuge tube with MM and group’s initials (I.S. D.K.)
- Add 125 microliters 5x GoTag Green buffer, 5 microliter forward and reverse primer each, and 105 microliter nuclease free water into MM tube. This is will be a master mix.
- Make sure to label 4, 0.5mL PCR tubes with sample and initials (I.S. D.K. and sample)
- Pipette 48 microliters of master mix into each of the 0.5mL PCR tube
- Into 2 of the 0.5mL PCR tubes, add 2 microliters od cDNA sample
- Into the other 2, add 2 microliters of water and label these as C for the control
- Concisely centrifuge tubes to pool liquid at the bottom.
- Make sure to secure the caps on the PCR tubes and place into thermocycler
- Store the samples at 20C
Conclusion: We didn’t measure anything in this lab, however we were introduced to a PCR technique which is used on cDNA to separate a gene of interest so it could be replicated and used for analysis. The second part of the lab was more focused on our group experiment.
Reflection: This lab experiment was very helpful it taught us how to do a PCR, prepare primers, and master mix and how to use thermocycler. For the group experiment, we were able to collect appropriate amount of sample tissue (tubes) and did dissections on one urchin from each group, total of 3 urchins.
Date: October 18th, 2011
Summary
In this lab we had an opportunity to do reverse transcriotion inorder to convert our sample of RNA, which we've collected during the previous lab experiment, to cDNA. Ultimatelly we stored tour contents on ice for later analysis. We have also produced an experimental and started setting everything up.
Reverse Transcription
Materials
- Gloves
- micropipettes
- Ice bucket
- Microcentrifuge tubes
- DI Water
- Thermal cycler
- RNA sample
- M-MLV Reverse Transcriptase
- M-MLV 5X Reaction buffer
- dNTPs
- Oligo dT
- 0.5 mL PCR tubes
Methods
- After removing the RNA from the ice, melt it and mix it thoroughly
- Label 0.5 mL PCR tube with your initials ( I.S. & L.T.) and "cDNA"
- Add 0.5 microliter RNA, 1 microliter oligo dT and 4 microliters of DI Water into this labeled 0.5 PCR tube
- Incubate the PCR tube for 5 minutes at 70 degrees in celsius insid ethe thermocycler
- After incubating, transfer the tube to ice bucket for 2 minutes (do this immediately)
- Vortex briefly to mix and desk top centrifuge it for about 5 seconds
- After the centriguge, add 5 microliter M-MLV 5X Reaction buffer, 5 microliter dNTP, 1 microliter M-MLV RT and 4 microliters of DI Water
- After the adding these contents, incubate it fro 60 seconds at 42 degrees celsius and after this process, heat inactivate for 3 minutes at 70 degrees in celsius.
- Centrifuge it again
- Place it on ice at -20 degrees in celsius
Primer Designe
We didn't have a chance to come up with any primers at that time, hopefully we will be better prepared for lab 4
Our goup's website: http://genefish.wikispaces.com/Green+Sea+Urchin+Predator+Response
Conclusion
We didn't really measure anything during this lab, we converted RNA into cDNA using reverse transcription.
Reflection
This lab was by far the best one we had so far. We learned more of the new molecular techniques such as how to convert RNA into cDNA by using reverse transcription. Another big accomplishment that we have done during this lab was setting up everything for our experiment which should begin on Thursday. The only thing that we still have to design is our primers, but other than that everything is going well.
Date: October 4th, 2011
Summary
In the first lab, we did a basic preparation for RNA extraction from a tissue, and protein extraction from a lung (really, lung?) -
Materials RNA Extraction:
1. C. gigas tissue 0.013g
2. gloves
3. 1.5 ml snap cap tube 10/4 HN AT
4. TriReagent
5.Vortex
6. Sterile pestle
7. Ice
8. Lab marker
Methods RNA Extraction:
1. Label the snap cap once the material is obtained
2. Transfer the tissue into the snap cap
3. Set it on the ice
4. Add 500 microliters of TriReagent to 1.5 ml tube containing the tissue.
5. Homogenized tissue reagent by using a sterile pestle
6. Add 500 microliters of TriReagent
7. Vortex sample 15 seconds
8. Store tissue at -80 C until next week
Materias Protein Extraction:
1. Gloves
2. Centrifuge
3. Bradford reagent
4. DI water
5. 1.5ml snap cap tube
6. 0.007g. Gigas natle tisse
7. 1000 microliter Cuvette
8. Sterile pestle
9. CellLytic MT solution
10. Screw cap tube
11. Spectrophotometer
Methods Protein Extraction:
1. Transferer C. gigus gill tissue into the snap cap
2. Added 500 microliters of CellLytic MT solution to the tube containing tissue sample
3. Homogenize tissue with sterile pestle
4. Place the sample in a refrigerator and microfuge for ten minutes
5. Transfer supernatant to new tube
6. Add 15 microliters of protein supertant to new 2ml tube that contains 15 microliters of DI
7. Add 30 microliters of DI water into the 2ml tube
8. Add 1.5 ml of Bradford Reagent to each of the 2ml tubes
9.Iincubate samples at room temperature for 10 minutes
10. Add 1mL of well mixed protein sample to a cuvette with a P1000 micropipette
11. Add 1mL of well mixed blank sample to a different cuvette
12. Zero spectrophotometer with blank
13. Record the protein sample absorbance value
14. Absorbance values were 0.111 and 0.114
15. Find the average of these two values and the concentration from given standard curve y=1013.9x. X is the absorbance. Multiply the value of average absorbance by 2 and the absorbance will be equal to0.228. The protein concetration is 231.17 micrograms per mL after all of the calculations
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Where are your results?
Conclusion
Based on our data/results, we can conclude that they are relatively reasonable: our absorbance from the sample was normal and we also had a reasonable protein concentration after carefully taking all the measurements.
Reflection
This lab introduced us to some of the new techniques that we have encountered and it also helped us to understand how to use methods like the bradford assay properly. From a scientific perspective, this experiment can be applied to multiple samples inorder to gain a better understanding of level of protein from various types of tissues. Protein is extremely important part of the organism and cell because of its function. Being able to quantify it can tell us a lot about specific tissue and organism. I would like to do a little research and see if our protein concentration of the tissue is similar to protein concentration of a different tissue but from the same species and how does it differs.
Date: October 11th, 2011
Summary: The purpose of this lab was to comlete the process of RNA. The homogenized mixture from the previous week was further processed by the addition of various chemicals including chloroform and isopropanol.
Materials RNA Extraction & Quantification
1. Vortex
2. Timer
3. Gloves
4. Protective eye wear
5. Micropipettes
6. Isopropanol
7. 75% EtOH
8. 0.1% DEPC Treated water
9. Ice
10. Phenol
11. Microcentrifuge tues
Method
1. Incubate RNA/TriReagent sample at room temperature for 5 minutes
2. Add 200 microliters of chloroform with a pipette (under the fume hood)
3. Vortex saple
4. Incubate the sample for another 5 minutes at room temperature
5. Microfuge in the refrigerator for 15 minutes
6. Separate the clear pipetted aqueous phase away from interphase and transfer the aqueous phase into a different tube labled AT/HN RNA 10/11
7. Dispose the liguid waste from the old tube
8. Add 500 microliters of isopropanol to the new tube that contains RNA, close and mix
9. Incubate it at room temperature for 10 minutes
10. Microfuge it for 10 minutes
11. Pipette the supenatant
12. Add 1000 microliters of 75% EtOH and dislodge pellet from bottom of the tube
13. Microfuge it at 7500 rpm for 5 minutes
14. Pipette the remaining EtOH
15. Air dry it at room temperature for 5 minutes
16. Add 100 microliters of 0.1% DEPC-H2O
17. If pellet is not dissolved incubate the sample at 55 degrees for 5 minutes
18. Place the sample on ice
19. Add 2 microliters of 0.1% DEPC-H2O onto nanodrop pedestal, press blank button on the computer
20. Wipe and then add2 microliters of our RNA solution to pedestal
21. Close the arm and click measure and put the sample on ice to store at -80C.
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Conclusion
The RNA concentration that we have acquired was 102 nanograms per microliter, which was a reasonable result. In comparison, however, other groups had much higher RNA concentrations and some had measurements of thousands. Pure RNA ratio for A260/280 should be 1.8-2.0 and pure RNA ratio for A260/230 should be 1.5-2.0. One of our ratios was within the normal range where as the other one was little below: A260/230 ratio of 1.87 and A260/280 ratio of 1.70. This slightly lower ratio for A260/280 indicates the absorbance of proteins.
Reflections: The purpose of this lab was to finish our RNA extraction that we have started a week ago and also to quantify the concentration and purity of our extracted RNA. Measurements of RNA and their purity can tell us a lot of information about gene expression in the tissue from which the RNA was extracted and physiological responses of organisms. By reflecting more on this lab, it was very useful because we were able to use numerous componds when preparing the tissue and I also wish I could learn more about the graph that we obtained on the computer monitor for our RNA ratio.