Charlie+Heller+Fish+441


 * Experiment- entry 2**
 * 11/24/2013**

11/19: We discovered that our specimens in the experimental treatments have been dying faster than anticipated, especially in the copper treatments. For this reason, we have decided to use the samples that we have collected from this day and on previous days (baseline sample and those from 11/12), and to not collect any more samples. Doing so could potentially provide us with exceptional data, so we have decided to avoid that risk.

Protein extraction was completed on all samples collected to date.

11/24: Protein quantification was completed using extracted samples from 11/19.

Western blot scheduled to take place on 11/26 and 27.


 * Experiment- entry 1**
 * 11/17/2013**

The purpose of my experiment is to compare the expression of HSP-70 in mussels to the concentration of copper, as well as lower pH levels. This will be done using four different treatments (one control, one with copper, one with lower pH, and one with both copper and low pH), with 10 mussels in each treatment. Each week, including the day of the commencement of the experiment, gill tissue samples will be extracted from an individual from each of the four control groups. These samples will be used in protein quantification and SDS-page procedures to determine HSP-70 expression in each treatment.


 * Lab #5**
 * 11/3/2013**

In last week's lab, we finalized the details of our overarching experiment and organized a final schedule for our collective experimental procedure. Unfortunately, the metallothionein team ran into some major obstacles, in that metallothioneins are related to antibodies in such a way that prevents us from performing PCR and/or western blots for this gene/protein. For this reason, we decided to change our observed mechanism to HSP-70, and how its function/concentration is affected by the concentration of cadmium in the environment. This will mean that my new primers will arrive somewhat late into the experiment, but I should be able to manage regardless.

---

The purpose of this lab was to practice using SDS-PAGE and Western blot techniques to extract and quantify proteins from our previous samples. This further illustrated the importance of the central dogma of biology, as well as helped prepare us for our lab experiments.
 * Lab #4**
 * 10/27/2013**
 * Summary:

Protein extraction: SDS-PAGE [] The blot on the far left is the ladder; the one on the far right was my personal sample. Protein extraction: Western Blot Immunodetection While no specific image was recorded for the results of the western blot, it’s safe to say the experiment was a success. HSP 70 was successfully proven to be present in the sample at expected quantities.
 * Materials:
 * Micropippettes (1-1000 µL)
 * Sterile filter pipette tips (1-1000 µL)
 * Sterile gel loading tips
 * 1.5 mL screw cap tubes
 * Microcentrifuge tube rack
 * Heating block w/ water bath
 * Protein gel box
 * SDS/PAGE gels
 * Gel loading tips
 * Tryas for straining gels
 * Power supply
 * 2X SDS reducing sample buffer
 * Protein ladder marker
 * Gel running buffer
 * Methods:
 * In a fresh 1.5 mL snap cap, added 15 µL of protein stock and 15 µL of the reducing sample buffer. Snap cap briefly centrifuged to pool liquid.
 * Sample boiled for 5 minutes, then centrifuged again for 1 min.
 * Entire sample added to one well, as well as one ladder for comparison. Lid placed on box, electrodes plugged in, and 150V run through sample for 45 minutes (see results section for picture)
 * Results:
 * Materials:
 * Nanopure water
 * gel staining tray
 * Blocking Solution
 * rotary shaker
 * Primary Antibody Solution
 * Antibody Wash
 * Secondary Antibody Solution
 * Chromogenic Substrate
 * SDS-PAGE gel
 * Tris-Glycine transfer buffer
 * filter paper
 * nitrocellulose membrane
 * semi-dry transfer station
 * Methods:
 * Blotting sandwitch assembled within a semi-dry blotting apparatus. Blot transferred for 30 minutes at 20 volts.
 * Gel removed from sandwitch and rinsed. Membrane placed in plastic block with 10 mL blocking solution. Blocking occurred for only 30 minutes.
 * Results:

The results of both the SDS-PAGE and Western blot were within expectations. I feel comfortable that I could replicate the techniques utilized in this lab in later attempts for my personal project. The purpose of this lab was primarily to give us an opportunity to practice protein extraction and quantification techniques, but also to help further illustrate the importance of the central dogma of biology. These techniques might be used in a study that would compare the abundance of a given protein relative to its partner mRNA and DNA. ---
 * Conclusion:
 * Reflection:
 * Lab #3: 10/15/2013**

Note: Two labs were performed today- one of qPCR, and one of protein extraction. The results, conclusions, and reflections of these labs will be recorded during Lab #4.

Part 1: qPCR Procedure The purpose of this lab was to help us learn the both the purpose and methods of qPCR. cDNA samples obtained from Lab #2 were used to quantify the presence of a certain gene of DNA.
 * Summary


 * Materials
 * PCR Plates (white) with optically clear caps
 * 1.5 mL microfuge tubes (RNAse free)
 * Nuclease Free water
 * Filter tips
 * Opticon thermal cycler
 * Kim wipes
 * 2x Immomix Master Mix
 * SYTO-13 Dye
 * Microfuge tube racks
 * Ice buckets
 * Timers
 * Upstream & downstream primers (DNMT)
 * cDNA samples (see Lab #2)
 * Methods
 * 50 μL Master mix (see Lab #2) was mixed with 2.0 μL of both upstream and downstream DNMT primers (for a total of 4.0 μL), as well as 42 μL of nuclease free water in a separate container. The volume of this new master mix was 96.0 μL.
 * The new master mix was evenly distributed between four PCR plates (24.0 μL of MM in each plate).
 * cDNA samples were thawed. Then, 1 μL of cDNA was added to two of the wells. 1 μL of nuclease free water was added to the other two wells. The former two wells were the experimental group, and the latter two were the control.
 * NOTE: It’s possible that I may have made mistakes at this step. I may have confused which plate was which while adding the cDNA and nuclease free water, which would explain why my results were inadequate.
 * The four wells were capped securely, and sent to undergo qPCR.
 * qPCR conditions:
 * 95°C for 10 minutes
 * 95°C for 15s
 * 55 °C for 15 s
 * 72°C for 15 s (+ plate read)
 * Return to the second bullet 39 more times
 * 95°C for 10s
 * Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

Part 2: Protein Extraction and Analysis The purpose of this lab was to give us an opportunity to practice protein extraction and quantification techniques. The quantification portion of this lab, which will be performed using a Bradford Assay, will be performed during Lab #4.
 * Summary
 * Materials
 * Micropipettes (1-1000uL)
 * Sterile filter pipette tips (1-1000uL)
 * Sterile (RNase free) 1.5mL microcentrifuge tubes
 * Sterile 2 mL screw cap microcentrifuge tubes
 * Sterile disposable pestles
 * Spectrophotometer
 * Cuvettes for spectrophotometer
 * Microcentrifuge (refrigerated) or in fridge
 * Ice buckets
 * CelLytic MT Cell Lysis Reagent (with Protease Inhibitor Cocktail added)
 * Coomassie Protein Assay Reagent
 * DI water
 * Tissue sample #84 (Gill tissue from Olympia Oyster)
 * Methods
 * Weight of tissue sample not recorded due to malfunctioning scale.
 * 500 μL CellLytic MT solution added to the 1.5 mL snap cap containing sample tissue. Sample was then homogenized to a satisfactory degree (i.e. until no large chunks of tissue could be seen) using a sterile disposable pestle.
 * Snap cap containing sample was closed and mixed inverted several times; then, sample was placed in a refrigerated microfuge and spun at maximum speed for 10 minutes.
 * Supernatant was extracted from the snap cap and transferred to a new tube. Tube containing supernatant (aka new sample) was then stored on ice.

---

The purpose of this lab was to continue to practice the methods of DNA and RNA extraction that we will be using in this class. Additionally, we were meant to learn the advantages of creating cDNA using extracted RNA. RNA was isolated using TriReagent, and used to create cDNA. Sample OLY 31g was used to produce the following results.
 * Lab #2: 10/13/2013**
 * Summary of the lab

Materials Methods: RNA extraction Methods: cDNA production NOTE: RNA quantification not performed at this time.
 * Micropipettes (1-1000 μL)
 * Sterile filter pipette tips (1-1000 μL)
 * 1.5 mL microcentrifuge tubes
 * Microcentrifuge tube rack
 * Ice buckets
 * Phenol/chloroform waste containers (liquid / solid)
 * Vortex
 * Hot water bath
 * Nanodrop spectrophotometer
 * Chloroform
 * RNase free water
 * Chloroform
 * Isopropanol
 * 75% ethanol
 * 0.1% DEPC treated water
 * Oligo dT
 * 1) Added 500 μL of TriReagent to homogenized tissue sample from previous lab. Incubated the sample at room temperature for 5 minutes.
 * 2) Added 200 μL of chloroform to sample under fume hood. Upmost safety precautions taken
 * 3) Sample vortexed vigorously for 30 seconds, then allowed to incubate for 5 minutes at room temperature.
 * 4) Sample spun in refrigerated microfuge for 15 minutes, at which time it was carefully removed. The aqueous phase was removed from the snap cap and transferred to a new one; the remaining interphase and solute were disposed of.
 * 5) 500 μL isopropanol added to new snap cap. Sample was mixed through repeated inversion, and then incubated at room temperature for 5 minutes.
 * 6) Sample was then spun at refrigerated microfuge for 8 minutes.
 * 7) Supernatant was removed from the tube, leaving only a small pellet. 1 mL 75% EtOH was added, and the pellet was dislodged from the tube via vortexing. The sample was then spun again in the refrigerated microfuge for 5 minutes.
 * 8) Supernatant carefully extracted from sample without disturbing pellet. Sample vortexed in order to pool remaining EtOH in order to ease further removal. Tube left open at room temperature for 5 minutes or until sample was dry.
 * 9) 100 μL 0.1% DEPC-H2O added to sample, and pipetted up and down until pellet dissolved.
 * 10) Tube incubated at 55°C for 5 minutes. Upon removal, sample flicked a few times (to further homogenize) and placed on ice.
 * 1) 5 μL RNA, 1 μL oligo dT, and 4 μLH2O added to a 0.5 mL tube and incubated at 70°C for 5 minutes.
 * 2) 14 μL of a mixture of DntDs, buffer solution, mmLv-RT and H2O added to sample. Sample then left on ice for 1 hour.

Since RNA quantification has not been performed on my sample as of yet, the quality of my RNA and cDNA is currently unknown. However, the procedure was followed with precision, and no mistakes were observed, leading me to believe that my samples are likely of reasonable quality.
 * Results

While the value of the purity of my sample is currently unknown, I would not be upset if it turned out poorly. My objective coming into this lab was to become confident using DNA/RNA extraction techniques, and I feel that I accomplished this extremely well. I feel fully confident in my ability to not only perform these procedures, but any variant thereof.
 * Conclusions

This lab was essentially the second half of Lab 1, and as such, its purpose was very similar: to enhance our comprehension of, and ability to complete RNA and DNA extractions. More importantly, it also further enhanced our understanding of the central dogma of biology- specifically, the importance of the roles that that these nucleic acids play in molecular biology. As with the previous lab, these methods would most likely be used to study gene expression, with a possible specific application in understanding and treating inheritable diseases. There isn’t much of the procedure that was unclear to me, but I would like for there to be more information on the specific real-world applications of these techniques.
 * Reflection
 * 441 Lab 1**
 * 10/1/2013**
 * 10/1/2013**

The purpose of this lab was to practice DNA and RNA extraction techniques, which will be used in this lab for the duration of this class. Samples of Pacific Oyster gill tissue were subjected to various controlled chemical and physical disturbances in order to concentrate 25-50mg of DNA, and 50-100 mg of RNA respectively.
 * Summary of the lab

Samples of Pacific Oyster gill tissue was used in both procedures. >>>
 * Materials and Methods
 * 1) Materials
 * 2) Micropipettes (1-1000 µL)
 * 3) Sterile filter pipette tips
 * 4) Sterile 1.5 mL microfuge tubes
 * 5) Sterile disposable pestles
 * 6) Vortex
 * 7) TriReagent
 * 8) Microcentrifuge & tube rack
 * 9) Razor blades
 * 10) DNazol
 * 11) 100% ethanol
 * 12) 75% ethanol
 * 13) 0.1% DEPC water
 * 14) Nanodrop
 * 15) Ice buckets
 * 16) Gloves
 * 17) Methods
 * 18) RNA isolation
 * 19) 500 µL of TriReagent added to the 1.5 mL snap cap containing RNA sample tissue.
 * 20) Sample homogenized using disposable pestle. Sample then subjected to vortex for a short amount of time. These two actions were repeated until a suitable level of homogenization was achieved.
 * 21) NOTE: Only half of the TriReagent prescribed by the procedure was used.
 * 22) Once sample was homogenized, it was stored at -80°C.
 * 1) DNA isolation
 * 2) Added 0.5 mL DNazol to the 1.5 mL snap cap containing DNA sample tissue. Sample was homogenized using a disposable pestle.
 * 3) Added another 0.5 mL DNazol to sample. Sample was then incubated for 5 minutes.
 * 4) Sample was then spun in a microcentrifuge at 10,000 g for 10 minutes, after which point all supernatant was extracted from the snap cap.
 * 5) Added 0.5 mL 100% ethanol to the sample. Sample was mixed by repeatedly inverting, then stored at room temperature for one minute, at which time ethanol was extracted. No cloudy precipitate formed by this point, so this step was repeated.
 * 6) Lysate was removed from the sample.
 * 7) 1 mL 75% ethanol was added to the sample via pipette. The sample was inverted several times and allowed to sit for 1 minute. This step was then repeated, after which point the remaining ethanol was removed via pipette.
 * 8) At this point, the remaining tissue sample had formed a relatively large lump. 300 µL 0.1% DEPC was added to the sample. The sample was then homogenized by alternating between using a sterile pestle and the vortex for roughly 30 minutes, which had little effect on the lump’s original integrity. At this point, the sample was brought to the Nanodrop to quantify.


 * Results
 * o 50.6 ng/µL
 * § 50.6 x 300 = 15180 ng
 * o λ: 230
 * o Abs.: 10.630
 * o A-260 10-mm path = 1.012
 * o A-280 10-mm path = 0.522
 * o 260/280 = 1.94
 * o 260/230 = 0.10

My results were slightly disappointing, in that the DNA concentration was unexpectedly dilute. The 260/280 ratio was just outside of acceptable bounds (1.7-1.9). Additionally, the 260/230 ratio was phenomenally lower than it should have been, which indicates that there was some serious contamination in my sample at some point. It’s most likely that this occurred right after step 3, when I was disposing of the supernatant- I believe I might have mixed up which part of the snap cap I was supposed to remove. While it could be argued that the quality of my DNA extraction was adequate considering this was my first attempt at extracting DNA, I feel that it would be in my best interest for me to practice this technique several more times before I can satisfactorily replicate it.
 * Conclusions

The purpose of this lab was two-fold: to guide us through our first attempts at DNA and RNA extraction, but more importantly, to help illustrate the central dogma of biology: that all complex life uses DNA, RNA, and proteins – the building blocks of life – to satisfy both the organism’s immediate and long-term needs. The procedures used in this lab are used to measure the concentration of DNA and RNA in a particular sample tissue. These techniques could be used to help detect hereditary diseases before they manifest, as well as to help understand the nature of such diseases. The only unclear component of this procedure to me was how repeatedly soaking the sample in ethanol wouldn’t damage the DNA strands beyond useful laboratory applications.
 * Reflection

Genes that I find particularly interesting include HOX and other related developmental genes.