yyl

=**November 29th, 2011**=

__**Reflection:**__


 * 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. **

//1) The restriction enzymes used in this lab may have cut in single nucleotide polymorphisms, which are abundant in oysters and would result in non-selective amplification.//

//2) I could have gotten all of the samples contaminated, which would result in similar amplification and band results in the gel.//


 * 2. What are two obstacles that you encountered during your lab work and experimental design? Did these obstacles affect your results? Why? **

//1) I couldn't find primers on NCBI that included a methylated site with CCGG for the amp-gigasin 2 gene, but Emma helped me design primers on another program so I was able to continue with PCR and the rest of my experiment.//

//2) I didn't see a pellet form during DNA extraction for most of my samples, but NanoDrop measurements of the DNA concentrations proved that DNA was sufficiently there so it did not affect my results.//


 * 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 research is important because it can teach future students about properly designing primers, or teach future scientists lessons about the amp-gigasin 2 gene and designing primers for amplification. I would tell the non-scientist that challenged the importance of this research that we can all make mistakes or discover unexpected results, and they may be more valuable than predicted results in solving how exactly anthropogenic environmental change affects oysters (important aquaculture organisms!).//


 * 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? **

//The fact that all of the wells in the electrophoresis gel show the same bands. Theoretically, HpaII is not able to cut methylated CpG sites and should show a strong band near the top of the wells in methylated genes. I can perform qPCR to figure out if the problem is the restriction enzymes cutting at single nucleotide polymorphisms or if I had contamination in each of the samples. user:emmatsqPCR is really just to improve the sensitivity of detection. The enzymes probably didn't cut all of the DNA and the coarse detection quality of gel electrophoresis could mean that even if some samples were methylated they still didn't look different.//


 * 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. **

//1) Ocean acidification can not only have detrimental effects on oyster calcification but on their metabolism as well. In addition to ocean acidification, the experiment also increases exposure temperature. Ocean acidification will weaken and narrow their thermal tolerance range.//

//2) Calcification rates decline linearly with decreasing pH. This experiment was performed taking saturation solubility products for calcite and aragonite in order to measure calcification rates. Their future endeavors include discovering potential adaptions to help oysters better survive increasing carbon dioxide levels in sea water.// //user:emmatswhat are the citations for these papers?//

=**November 22nd, 2011**=

__**Summary:**__ To work on individual projects. Mine is to investigate DNA methylation on the amp-gigasin 2 gene using restriction enzymes, PCR, and gel electrophoresis. = = __**Methods:**__ //Restriction Enzymes Digestion// //-// Use the NanoDrop spectrophotometer to determine DNA concentrations in ng/µL - Calculate µL needed to equal **1µg** DNA, then add Nanopure H2O to equal **44µL** for HpaII digests, **44.5µL** for MspI digests, and **50µL** for negative controls. - For HpaII digests, add **5µL** of #1 buffer and **1µL** of enzyme, for MspI digests, add **5µL** of #4 buffer and **0.5µL** of enzyme - Incubate both digests and controls at 37°C overnight, heat stop 20min at 65°C for HpaII and 80°C for MspI

//PCR, Gel Electrophoresis// - Procedures can be found in 10/25/11 and 11/1/11 entries.

__**Results**__ //Observations// - Heating block for heat stopping takes a long time to heat from 65 to 80 degrees

//Deviations// - Used 2x GoTaq master mix again - Used 1.5g of agarose instead of 2g, 100mL of TAE buffer

//Calculations// - Digest DNA amounts
 * **Ploidy** || **Weight (g)** || **μL equal to 1μg** |||||| **μL Water to add** ||
 * ||  ||   || //hpa// || //msp// || //control// ||
 * 3n || 47 || 101 || 0 || 0 || 0 ||
 * 3n || 25 || 5.52 || 38.48 || 38.98 || 44.48 ||
 * 3n || 30 || 14.84 || 29.16 || 29.66 || 35.16 ||
 * 3n || 35 || 5.63 || 38.37 || 38.87 || 44.37 ||
 * 3n || 37 || 8.55 || 35.45 || 35.95 || 41.45 ||
 * 2n || 45 || 6.29 || 37.71 || 38.21 || 43.71 ||
 * 2n || 55 || 9.35 || 34.65 || 35.15 || 40.65 ||
 * 2n || 47 || 6.46 || 37.54 || 38.04 || 43.54 ||
 * 2n || 53 || 11.47 || 32.53 || 33.03 || 38.53 ||
 * 2n || 48 || 8.61 || 35.39 || 35.89 || 41.39 ||

//Electrophoresis// Samples in well layout is detailed in chart below
 * |||||||||||||||||||||||||||||| **Diploid** ||  ||
 * //well 1// |||||||||| //MspI// |||||||||| //Control// |||||||||| //HpaII// || //well 20// ||
 * **Ladder** || 53 || 45 || 55 || 48 || 47 || 48 || 53 || 45 || 55 || 47 || 48 || 53 || 55 || 45 || 47 || **Ladder** ||
 * |||||||||||||||||||||||||||||| **Triploid** ||  ||
 * //well 21// |||||||||| //MspI// |||||||||| //Control// |||||||||| //HpaII// || //well 30// ||
 * **Ladder** || 35 || 47 || 37 || 30 || 25 || 47 || 37 || 35 || 25 || 30 || 37 || 35 || 30 || 25 || 47 || **Ladder** ||
 * **Ladder** || 35 || 47 || 37 || 30 || 25 || 47 || 37 || 35 || 25 || 30 || 37 || 35 || 30 || 25 || 47 || **Ladder** ||

__**Conclusions**__ What I expected was there to be more DNA methylation in triploids than diploids, which translates to longer separation of fragments in the lower row vs. top row in the gel in the Msp columns. HpaII does not cut methylated CpGs, so there should be a concentration of DNA in the Hpa columns, which is not seen in the gel. Perhaps a qPCR run of both enzymes can solve the mystery of all columns showing the same band patterns (except the DNA ladder).

__**Reflection**__ The purpose of this lab was to further investigate our research proposal. I performed PCR and gel electrophoresis in order to measure and compare DNA methylation of the amp-gigasin 2 gene between triploid and diploid oysters, which can be used for conservation studies and agriculture sustainability in the face of ocean acidification. There is nothing unclear about the given procedures.

=**November 15th, 2011**=

__**Summary:**__ To measure methylation using cytosine methylation dot blots, and using qPCR to measure gene expression.

// DNA Dilutions // - Dilution DNA to 50ng/µL in a 40µL total volume. - Prepare dilutions: //Dot Blot// - Cut nylon membrane to fit 72 wells of manifold, cover nylon membrane in 6X SSC for **10min**. Put manifold with the membrane lying on top of the filter paper. - Denature DNA in boiling water for **10min**, then transfer to ice. Turn on vacuum, add **500****µL** of 6X SSC to each well, let SSC to flow through. - Adjust vacuum speed so the SSC to filter through takes a few min. Spin down DNA for 5 min. - Add all of the DNA to each well, not touching membrane, recording DNA names and locations. Allow samples to flow through. - Soak filter paper cut to size in denaturation buffer, then remove manifold and transfer membrane (dot side up) to filter paper soaked in denaturation buffer. - Wait 5min, place membranes on dry filter paper and dry. Wrap dried blot in plastic wrap and place DNA-side-down on UV transluminator for **2min** at 120kJ.
 * __Methods:__**
 * = ** Dilution ** ||= ** TARGET **
 * amount ** ||= ** ul of H20 ** ||= ** ul of 20X SSC ** ||= ** ul of 50ng/ul **
 * DNA sample ** ||
 * 1 || 800 ng || 124 || 60 || 16 ||
 * 2 || 400 ng || 132 || 60 || 8 ||
 * 3 || 200 ng || 136 || 60 || 4 ||
 * 4 || 100 ng || 138 || 60 || 2 ||
 * 5 || 50 ng || 139 || 60 || 1 ||

//Chromogenic Immunodetection// - Prepare **20mL** of Blocking Solution: Ultra filtered Water **14mL**, Blocker/Diluent (Part A) **4mL**, Blocker/Diluent (Part B) **2mL** - Place membrane in **10mL** of Blocking Solution in a covered, plastic dish, incubate **30min** on a rotary shaker @ 1 **revolution/sec**. - Decant Blocking Solution, rinse membrane **5min** with 20mL H2O, then decant. (2x) - Prepare **10mL** of Primary Antibody Solution (1:5000 dilution): Blocking Solution **10mL**, 5-MeC antibody **2µL** - Incubate membrane 1hr with **10mL** Primary Antibody Solution. - Decant primary antibody and wash the membrane 5min with **20m**L of 1X TBS-T, decant. (3x) - Incubate membrane 30min in **10mL** secondary antibody solution, decant. - Wash membrane 5min with **20mL** TBS-T, decant (3x) - Rinse membrane 2min with **20mL** H2O, decant. (2x) - Incubate membrane (1-60min) in **5mL** Chromogenic Substrate until color develops - Rinse membrane 2min with **20mL** H2O, decant. (2x) - Dry membrane of clean piece of filter paper

//qPCR// - Thaw cDNA samples.

- Prepare master mix, **25μL** reaction volu me: - Add **25μL** master mix to wells of a white PCR plate, and ** 2 μ** **L** cDNA template to each reaction. (add ultra pure water for negative controls), cap well. - Place strips on ice, load plate, use the following conditions:
 * **Component** || **Volume** || **Final Conc.** ||
 * Master Mix, 2X (Immomix) || 12.5µL || 1x ||
 * Syto-13 dye (50uM) || 1µL || 2µM ||
 * upstream primer, 10μM || 1.25μl || 2.5μM ||
 * downstream primer, 10μM || 1.25μl || 2.5μM ||
 * Ultra Pure Water || 7uL || NA ||

1. 95°C for 10 minutes 2. 95°C for 15s 3. 55 °C for 15 s 4. 72°C for 30 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)

__**Measurements**__ //Nanodrop DNA measurements// __**Results**__ //Observations// - First trial of 6X SSC flow through was too fast, use slower settings.
 * **Ploidy** || **Weight(g)** || **260/280** || **260/230** || **ng/****μL** ||
 * Triploid || 47 || 2.24 || 0.03 || 10.1 ||
 * Triploid || 25 || 1.85 || 0.33 || 181.0 ||
 * Triploid || 30 || 1.98 || 0.21 || 67.4 ||
 * Triploid || 35 || 1.86 || 0.42 || 177.6 ||
 * Triploid || 37 || 1.87 || 0.29 || 116.9 ||
 * Diploid || 45 || 1.71 || 0.30 || 158.9 ||
 * Diploid || 55 || 1.85 || 0.19 || 107.0 ||
 * Diploid || 47 || 1.71 || 0.29 || 154.9 ||
 * Diploid || 53 || 1.79 || 0.16 || 87.2 ||
 * Diploid || 48 || 1.98 || 0.21 || 116.1 ||

//Deviations// - Crosslinker used instead of UV transilluminator

//Calculations// DNA dot blot dilutions: //-// Diploid 48; 116.1ng/μL. CV = C2V2, V = C2V2/C, V = (40μL*50ng/μL)/(116.1ng/μL) = **17.2μL DNA**, 40μL total - 17.2μL DNA = **22.8μL H2O** for initial dilution

__**Conclusions**__ Did not have time to finish within a 3hr lab period. Everything else in the procedure performed went well. I will strive to be more efficient in executing actions in the future.

__**Reflection**__ The purpose of this lab was to perform dot blotting and qPCR. The procedures were performed to examine methylation as well as gene expression, in hopes of gaining a better understanding of how organisms respond to stressors in their environment (such as our acidic water oysters). The instructions were very clear.

=**November 8th, 2011**=

__**Summary:**__ To perform DNA extraction on ocean acidification sample oysters using DNAzol.

//DNA extraction// - Homogenize tissue in ** 0.5mL ** of DNazol in 1.5mL microcentrifuge tube (MCT). Add additional ** 0.5mL ** DNAzol and mix. - Incubate **5min** @ room temperature (RT). Spin sample ** 10min ** @ 10,000g RT - Transfer supernatant to a new, labeled tube, add **0.5mL** 100% ethanol (EtOH) to sample. Invert tube 5-8x to mix, incubate 1min @ RT - Remove cloudy precipitate DNA and pipet into new tube, incubate 1min @ RT, remove excess liquid. - Pipette **1mL** 75% EtOH into DNA tube, invert 6 times, incubate 1min @ RT. Remove ethanol. (2X) - Add **300µL** 0.1% DEPC H2O to DNA. Pipette to mix and dissolve. Insert in NanoDrop machine to quantify.
 * __Materials & Methods:__**

//Measurements// (weight in mg)
 * Diploid || Triploid ||
 * 47 || 37 ||
 * 45 || 30 ||
 * 48 || 47 ||
 * 55 || 25 ||
 * 53 || 25 ||

__**Results:**__ //Observations// - During "remove cloudy precipitate", DNA did not remain a precipitate

//Deviations// - Did not add additional 0.5mL DNAzol after first addition. Added 2.4µL ProK and incubated 1hr @ RT - Added centrifuge step before removing cloudy precipitate DNA: centrifuge 5min @ 5,000g

//Calculations:// - N/A

__ Conclusions: __
Extraction was difficult. After centrifuging sample 5min @ 5,000g, very little trace of DNA pellets were present in tubes. I expected there to be pellets, but I suspect it could be that DNAzol and ProK did not have enough time to properly extract DNA. I will strive to do a better job in reducing contamination and improving DNA extractions.

__ Reflection: __
The purpose of the lab was to learn how to extract DNA. The procedures were used to examine gene expression at the DNA level, or prepare for qPCR, which may be used to study gene methylation in specimens in response to a change in their environment. The instructions were not vague, but it would have been better to explain more about ProK.

=**November 1st, 2011**=

__Summary:__
To run PCR through gel electrophoresis, extract RNA and perform SDS-PAGE and Western blots.

__Materials and Methods:__
//Agarose gel// - Add 2g agarose and 150mL 1x TAE into a 1L flask, microwave ~3 min. - Cool to RT, add 12uL 1.5% ethidium bromide (EtBr), mix well and pour into gel tray. - Add gel combs, wait for gel to set, wrap in plastic wrap, place gel in fridge.

//Electrophoresis// - Remove combs from wells, place gel in gel box, submerge with 1x TAE buffer - Load 7uL 100bp ladder in 1st well, 25uL PCR samples in the other wells - Run gel at ~ 100V for ~ 1hr, view gel on a UV transilluminator

//SDS-PAGE// - Boil water on hot plate. - Add 15uL protein stock to 15uL 2X Reducing Sample Buffer in 1.5mL screw cap tube (SCT), return protein stock to -20C freezer - Mix sample, centrifuge 10s, boil 5min - Load into well. Run 45min @ 150V. Trim wells at top of gel, notch a corner for orientation.

//Western Blot// - Soak filter paper, membrane and gel in Tris-Glycine Transfer Buffer 15min - Put the following blotting sandwich in blotting apparatus in order: anode, filter, membrane, gel, filter, cathode. - Transfer blot for 30min @ 20V, remove and rise gel with transfer buffer - Wash membrane twice, 20 mL H2O ea, 5min ea. Place membrane in box, add 10mL blocking solution - Incubate overnight on rotary shaker @ 1 rev/s - Decant liquid, rise membrane 5min with 20mL H2O, decant (2x) - Incubate membrane in Primary Antibody Solution (PAS), decant. Rinse with 20mL Antibody Wash (AW), decant. (3x) - Incubate membrane 30min in 10 mL of Secondary Antibody Solution (SAS), decant - Wash membrane for 5min with 20 mL of AW, decant. (3x) - Rinse membrane 2min with 20mL H2O, decant. (2x) - Incubate membrane in 5mL of Chromogenic Substrate (CS) until a purple band appears. - Dry membrane on clean piece of filter paper in air

__ Results: __
//Measurements// - 0.04g sample "protein, 2n wet mantle, YYL"

//Above left: DNA ladder used. Above middle: PCR gel under UV illumination. Shows contamination in wells with negative controls, primer dimers, and multiple products in some wells. Above right: Western blot. Nothing present = no HSP70 expression.//

//Left: layout of SDS page samples. Left: layout of PCR samples. Above right: SDS-page results. Dark bands = high protein concentration.//

//Observations// - Electrophoresis: strong band and faint band in both samples. Bands in controls - SDS-PAGE is running really fast after 15min.

//Deviations:// - Our TA already prepared the agarose gel - Only used 5uL of the DNA ladder for PCR - Ended SDS-PAGE after running for 25min

//Calculations://
 * N/A

__ Conclusions: __
Both our negative controls in the gel electrophoresis showed a band, which means contamination. Our samples had a dim band, which means formation of primer-dimers. I did not expect to see bands in the negative controls since the negative controls were not supposed to have DNA. We will strive to reduce contamination, to be able trust the future results of our sample PCR.

__ Reflection: __
The purpose of the lab was to learn how to run PCR products through gel electrophoresis, protein products through SDS-PAGE and Western blots. The procedures were used to visualize products separated by size, which may be used to study gene and protein expression in specimens in response to a change in their environment. The instructions were very clear.

=**October 25th, 2011**= >>
 * __Summary__
 * To dissect tissue and perform a PCR on cDNA with primers and Promega's GoTaq mix.
 * __Materials and Methods__
 * //Dissection//
 * See 10/18/11 instructions
 * //End-point PCR//
 * Label a **1.5mL** microcentrifuge tube "MM" (master mix) for //each// PCR reaction (rxn) and follow the recipe below:
 * Reagent || 1.reaction || x.reactions ||
 * 5x GoTaq Green buffer || 10 µL ||  ||
 * 10mM dNTP mix || 1 µL ||  ||
 * 10 µM forward primer || 1 µL ||  ||
 * 10 µM reverse primer || 1 µL ||  ||
 * GoTaq polymerase || 0.25 µL ||  ||
 * nuclease-free water || 36.75 ||  ||
 * Aliquot **48µL** of the MM in to each 0.5 mL PCR tubes, labeled with initials and sample. Add **2µL** of the sample and pipet to mix. Spin tubes and load into thermocycler according to the below instructions: follow the recipe below:
 * Step || Temperature || Time || Cycles ||
 * Denaturation || 95C || 5 min || 1 ||
 * Denaturation || 95C || 30 sec || 40 ||
 * Annealing || 55C || 30 sec ||^  ||
 * Extension || 72C || 90 sec ||^  ||
 * Final extension || 72C || 3 min || 1 ||
 * Hold || 4C || ∞ || 1 ||
 * //Samples://
 * cDNA stock: labeled "YM, 10/18"
 * 5x triploid wet oyster samples: "YYL, 3n, wet, mantle"
 * 5x triploid dry oyster samples: "YYL, 3n, dry, mantle"
 * __Results__
 * //Observations//
 * It was very difficult to shuck the oysters
 * //Deviations://
 * Used 2x GoTaq buffer instead of 5x
 * Did not add dNTPs and polymerases into master mix


 * __Conclusions__
 * No measurements were taken. Now that we are acquainted with the methods of PCR, we will be able to observe gene expression through amplification of our project samples and determine how environmental stressors affect the amplification.

>> =**October 18th, 2011**= >> >>>
 * __Reflection__
 * The purpose of the lab was to continue performing PCR on the cDNA stock produced 10/18/11 as well as extract samples for our group projects. The PCR procedures were used to measure gene expression, which may be used to study gene regulation in specimens in response to a change in their environment. What was unclear about the deviations was why dNTPs and polymerase were not added?
 * __ Summary __
 * To learn about and perform reverse transcribing RNA to cDNA using oligo dT and M-MLV reverse transcriptase, dissecting animals using the sterile technique, and designing and ordering primers.
 * __ Materials and Methods __
 * //Reverse transcriptase//
 * label a 0.5mL tube "cDNA", and add **5µL** of well-mixed stock RNA, **1µL** of oligo dT, and **4µL** of nuclease-free water (NFW). Incubate at **70°C** for **5min** and put on ice
 * centrifuge briefly and add **5µL** M-MLV 5X Reaction Buffer, **5µL** dNTPs, **1µL** M-MLV RT, and **4µL** NFW. Incubate at **42°C** for **1 hour** (hr) then at **70°C** for **3min**. Spin on desk centrifuge and store at **-20°C**
 * //Dissection//
 * shuck oyster at hinge using a shucker and follow cutting through adductor muscle. Open oyster and take care to keep body whole. Sterilize forceps with bleach and ethanol, and remove desired tissue to place into labeled tube.
 * dispose of unwanted tissue in designated locations and re-sterilize all materials.
 * //Primer design//
 * use NCBI primer and Primer 3 programs to design primers about 18-30 bases in length, has **<2°C** melting temperature difference, avoiding primer dimers, primer hairpins, high G/C repeats but has a G/C clamp at the 3' end.
 * //Sample//
 * "YM, 10/18/11, cDNA" in 0.5mL tube
 * __ Results __
 * //Measurements//
 * N/A
 * //Observations//
 * solution in 0.5mL tube was clear after every addition in the methods
 * //Deviations://
 * vortexed and spun the tube before incubating at both 70C and 42C.
 * did not perform the "dissection" section of the methods
 * //Calculations://
 * N/A


 * __ Conclusions __
 * We have not measured any results yet.

= = =**October 11th, 2011**= >> >>>
 * __ Reflection __
 * The purpose of the lab was to learn how to reverse transcribe RNA. The procedures were used to create cDNA from RNA, which may be used to study (through PCR) gene expression in specimens in response to a change in their environment. There is nothing unclear about the procedure, as it was very concise.
 * __ Summary __
 * The process of extracting RNA isolated by TRI-reagent from //Crassostrea gigas// (//C. gigas//) tissue is continued, and the resulting stock RNA sample is quantified using a Nanodrop spectrophotometer.
 * __ Materials and Methods __
 * //RNA Extraction//
 * Turn a heating block to **55°C** and incubate the homogenized tissue sample at RT for 5min. Add **200µL** chloroform, vortex for **30s**, then incubate at RT for **5min**.
 * Centrifuge at 0°C for **15min**, and transfer the top aqueous layer to a new micro centrifuge tube labeled "RNA". Add **500µL** isopropanol to the RNA tube, invert until solution is homogenous, and incubate at RT for **10min**.
 * Centrifuge at 0°C for **8min** and remove supernatant, leaving the white pellet. Add **1mL** 75% ethanol (EtOH) and vortex to dislodge pellet. Centrifuge at 7500g for **5min** and remove supernatant. Centrifuge **15s** and remove remaining EtOH.
 * Uncap tube and dry the pellet at RT for <5min, add **100µL** of 0.1% Diethylpyrocarbonate treated water ( DEPC-H2O) and pipet until pellet dissolves. Incubate at **55°C** for 5min, flick to mix and put on ice.
 * //RNA Quantification//
 * Use **2µL** of 0.1%DEPC-H20 on the Nanodrop pedestal, lower arm and set as blank. Pipet **2µL** of RNA sample onto the pedestal, lower arm and press "measure". Record the concentration, A260/280 ratio, and A260/230 ratio, and wipe Nanodrop arm using a KimWipe. Store "RNA" tube at **-80°C.**
 * //Sample://
 * //C. gigas// gonad tissue: labeled "YL, 10/4, gonad tissue" 0.031g
 * //C. gigas// gonad RNA: labeled "YM, 10/11, RNA"
 * __ Results __
 * //Measurements//
 * A260/280 ratio: 1.96
 * A260/230 ratio: 1.83
 * RNA concentration: 882.8 ng/µL
 * //Observations//
 * After the addition of chloroform and vortexing for 30s, the milky emulsion was a light pink colour.
 * After the addition of isopropanol and inverting, the solution appears uniform, and almost clear (but still slightly translucent)
 * //Deviations://
 * Some aqueous phase was left to prevent accidental transfer of the middle interphase layer below the top aqueous layer.
 * Did not vortex after addition of 1mL of ethanol
 * //Calculations://
 * Performed by the Nanodrop spectrophotometer using the Beer-Lambert law to calculate RNA concentration


 * __ Conclusions __
 * A typical A260/280 ratio should be ~1.8-2.0 and an A260/230 ratio ~1.5-2.0, both of which our measured values agree with. This means that our RNA was pure, since the measured values fall between the range of "pure" samples. Now that we are acquainted with the methods of quantifying RNA, we will be able to quantify our project samples after determining environmental stressors and measuring its affects on RNA concentration.

= = =**October 4th, 2011**= >> >>>
 * __ Reflection __
 * The purpose of the lab was to learn how to extract and quantify RNA from specimen tissue using the Nanodrop spectrophotometer. The procedures were used to measure RNA concentration, which may be used to study gene regulation in specimens in response to a change in their environment. There is nothing unclear about the procedure, as it was very detailed.
 * __ Summary __
 * RNA was isolated from //Crassostrea gigas// (//C. gigas//) gonad tissue using TRI-reagent. Protein was isolated from //C. gigas// mantle tissue using CellLytic MT, and the protein concentration was determined using the Bradford protein assay and a spectrophotometer.
 * __ Materials and Methods __
 * RNA Isolation
 * Lyse gonad tissue in 1.5milliliter (mL) snap-cap tube with 500microliter (uL) of TRI-reagent, and homogenize with disposable pestle. Add **500uL** additional TRI-reagent, vortex for 15 seconds (s), and store at -80ºC.
 * Protein extraction
 * Record weight of sample, lyse mantle tissue in 1.5mL snap-cap tube with **500uL** of CellLytic MT, and homogenize with disposable pestle. Close cap and invert until mixed. Centrifuge at maximum speed for 10 minutes (min), and store supernatant on ice. At what temp was the centrifuge? user:emmats
 * Protein concentration
 * Transfer **15uL** of the supernatant into a 2mL tube labeled "protein, BA" (for Bradford Assay) and dilute it with **15uL** de-ionized H2O (a 1:2 dilution). Add **30uL** of H2O into another 2mL tube labeled "blank. Then add **1.5mL** of BA reagent to both tubes, invert until mixed, and incubate at room temperature (RT) for 10 min.
 * Pipet **1000uL** of the blank solution to a disposable cuvette. Wipe the non-rough side of the cuvette with a Kim wipe and measure the absorbance at 595 nanometers (nm). Transfer **1000uL** of protein BA solution into a new cuvette and read the absorbance twice at 595 nm. Average the readings, and calculate using the y= 1013.9x equation and x= averaged absorbance to find the protein concentration.
 * Multiply the y by 2 to count for the 1:2 dilution, record the value. Store the protein supernatant from "protein extraction" in -20ºC.
 * Samples
 * // C. gigas // gonad tissue: labeled "YL, 10/4, gonad tissue" __0.031g__
 * // C. gigas // mantle tissue: labeled "YL, 10/4, mantle tissue". __0.008g__
 * Observations
 * Both the gonad and mantle tissues were difficult to completely homogenize using the disposable pestle.
 * Everything else in the procedure went according to plan.
 * Deviations
 * Instead of using a 2mL screw-cap tube, we used a regular snap-cap tube for the "protein concentration" procedure.
 * Calculations
 * [[image:Screen_Shot_2011-10-08_at_10.44.00_AM.png width="257" height="202"]]
 * Concentration was found using the **y= 1013.9x** equation above, and x= 0.479, our absorbance value, and was multiplied by 2 to correct for the 1:2 dilution used in the methods. y= 1013.9(0.479)*2= __971.3162 ug/mL__
 * __ Results __
 * Blank absorbance: 0
 * // C. gigas // mantle tissue protein absorbances: 478 nm, 480 nm, average= __479 nm__ absorbances are not measured in nm and these seem like very large numbers for absorbances (should be <1). The "nm" refers to the wavelength at which you measure the absorbance. user:emmats
 * // C. gigas // mantle tissue protein concentration: __971.3162 ug/mL__


 * __ Conclusion __
 * I didn't really expect any particular results, and the absorbance falling somewhere along the curve assures that we followed the procedure correctly. We will use these newfound skills to research protein concentration in specimens to determine their response to their environments.


 * __ Reflection __
 * The purpose of this lab was to learn how to extract RNA and proteins from specimen tissue. The procedures performed was used to measure protein concentration, which may be used to study gene regulation in specimens in response to a change in their environment. There is nothing unclear about the procedure, and everything was explained very clearly on the lab website.