Miranda's+Notebook


 * Date: Sunday, December 4th 2011**
 * Lab 8 Questions**

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.

HSP 70 gene expression was higher in the experimental groups then the control groups; this was to be expected. However, expression was slightly higher in the predator only tank then the synergistic tank. This could be due to a small sample size. user:emmatsExplain more! Why was it expected?

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

One obstacle was that we planned on tracking the urchin's shell-covering behavior during the period of the experiment. However, we were not able to view behavior at night due to poor light quality. This does not affect results in a dramatic way, because this is a small part of our experiment. We might only be able to compare behavior during daylight hours, however. Another obstacle was obtaining enough tissue to complete the DNA extraction; tube feet are extremely small and DNA extraction was low quality. However, this did not affect my results because I was able to obtain usable definitive 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 shows that HSP70 gene expression is in fact expressed in elevated levels in tanks involving a predator (versus a control). I would tell a non-scientist that challenged my research that this is yet another stress-induced aspect that triggers HSP70. Organisms experience a variety of stressors (ex. predator and salinity changes) on a daily basis, all of which have important implications on the 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?

I was completely stumped regarding why the urchins exposed to the predator alone expressed the HSP70 gene less then those exposed to both the predator and low salinity. For now, this is a mystery (however further research will be conducted on the matter). This could possibly be a fluke however, due to a small sample size.

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

Dumont: This paper explains the covering behavior that sea urchins exhibit. The article touches on various factors that could trigger the response, such as surge protection. It compares juvenile and adult urchins, noting that juveniles cover themselves more than adults. This is likely because juveniles are smaller and more vulnerable than adults, causing them to require more protection in order to survive.

Geraci: This paper examines HSP70 specifically in sea urchins. It will work well in describing gene expression of HSP70 in our control group, as it looks at this specifically. HSP70 is shown to be present in all life stages of the green sea urchin.

__Summary__ Purpose: Measure HSP70 gene expression Techniques: qPCR Samples: Strongylocentrotus purpatus
 * Date: Tuesday, November 22nd 2011**
 * Lab 7: qPCR for individual projects**

__Materials/Methods__ PREP: MASTER MIX AND WELL STRIPS 1. Master mix was created using the following concentrations:


 * ~ Component || Volume(uL) || Multiplier || TotalVolume(uL) ||
 * ~ x2 Immomix || 12.5 || x46 || 575 ||
 * ~ Syto-13 dye (50uM) || 1 || x46 || 46 ||
 * ~ Up Stream Primer || 1.25 || x46 || 57.5 ||
 * ~ Down Stream Primer || 1.25 || x46 || 57.5 ||
 * ~ Di H2O || 7 || x46 || 322 ||

2. Sample number was calculated: 20 urchins*2 for duplicates + 2 negative controls + 2 for error = 44 samples 3. 6 well strips were placed on mount and labeled with strip number and initials. 4. Vortex master mix for about 30 seconds or until thoroughly mixed 5. 23ul of master mix was placed in each well (Chart below lists sample location)


 * ~ Strip Number ||~ Well#1 ||~ 2 ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||
 * ~ 1 || Neg Control || Neg Control || C1 || C1 || C2 || C2 || C3 || C3 ||
 * ~ 2 || C4 || C4 || C5 || C5 || C6 || C6 || P1 || P1 ||
 * ~ 3 || P2 || P2 || P3 || P3 || P4 || P4 || P5 || P5 ||
 * ~ 4 || P6 || P6 || P7 || P7 || S1 || S1 || S2 || S2 ||
 * ~ 5 || S3 || S3 || S4 || S4 || S5 || S5 || S6 || S6 ||
 * ~ 6 || S7 || S7 || blank || blank || blank || blank || blank || blank ||

6. 2ul of DNA was added to urchin samples, 2ul of DI H2O was added to negative controls 7. Caps were placed on well strips

qPCR CONDITIONS 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

__Results/Conclusion__ Results will be obtained after thanksgiving break, however next I will begin to analyze data and write research paper.

__Reflection__ During this lab, I was able to complete the experimental portion of the project. All that is left is to analyze results and write the paper!


 * Date: Tuesday, November 15th 2011**
 * Lab 7: qPCR, DNA extraction**

__Summary__ Purpose: Measure methylation using dot blot, preform qPCR

__Materials/Methods__

DNA DILUTIONS 1. Obtain DNA sample 2. Dilute DNA so that you have 50 ng/µL in a total volume of 40 µL 3. Label 5 1.5ml snap cap tubes with initials, DNA, and target concentration 4. Prepare five dilutions with the following target concentrations (200ul total volume): 5. Set DNA aside to observe dot blot manifold
 * = ** 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 ||

DOT BLOT 1. Cut nylon membrane to fit 72 wells 2. Soak membrane in 6X SSC (enough to cover) for 10 min 3. Cut filter paper to size of membrane 4. Assemble manifold with membrane on top of filter paper 5. Denature DNA in boiling water for 10min 6. Transfer DNA to ice immediately 7. Switch on vacuum. Apply 500ul of 6X SSC to each well and allow SSC to filter through. Adjust vacuum speed so that it takes a couple of minutes for the SSC to filter through. 8. Spin down DNA for 5 min 9. Apply entire volume of DNA to wells without touching membrane 10. Note where you have applied your samples and each samples concentration on the blot 11. Allow samples to filter through. 12. Soak filter paper cut to size in denaturation buffer 13. Once filtered through, dismantle manifold and transfer membrane (dot side up) to filter paper soaked in denaturation buffer and let sit for 5m 14. Place membranes on dry filter paper and let dry 15. Wrap dryed blot in plastic wrap and place DNA-side-down on UV transluminator for 2 mins at 120kJ. This immobilizes the DNA

qPCR 1. Prepare master mix:

//For a 25μl reaction volu//me:
 * **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 ||

2. Add mastermix to wells of a white PCR plate 3. Thaw cDNA samples. 4. Add 2uL cDNA template to each reaction. 5. Add 2uL of ultra pure water to the negative control wells. 6. Cap the wells 7. Spin the strips to collect volume in the bottom of the wells. 8. Ensure the lids are clean and place strips on ice. 9. Load the plate, verify the PCR conditions and start the run

PCR conditions: 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)

__Results/Conclusion__ Primers were correct, however gel showed primer dimer.

__Reflection__ In this lab, I learned how to preform qPCR and dot blotting.


 * Date: Thursday, November 10th 2011**
 * Lab 6 Continued: Reverse Transcription**

__Summary__ Purpose: Transcribe sea urchin RNA to cDNA Samples: Taken from Strongylocentrotus droebachiensis

__Materials/Methods__

1. Invert samples several times 2. Label 20 0.5ul PCR tubes with samples (C1-6, P1-7, S1-7) 3. Add 2ug of RNA to each tube 4. Add 1ul of oligo dT to each tube 5. Incubate samples for 5min @RT 6. Prepare Master Mix: In a 1.5ml tube combine the following: 7. Add 11ul of master mix to each sample 8. Incubate samples in thermocycler: x1- 90C for 5 min,x40 cycles of (90C for 15s, 55C for 15s, 72C for 15s), x1- 71 for 5 mins. 9.Spin down samples in desk top centrifuge 10. Store at -20C
 * Name || Amount(uL) ||
 * M-MLV 5X Reaction Buffer || 5 * 21 = 105ul ||
 * dNTPs || 5* 21 = 105ul ||
 * M-MLV RT || 21ul ||

__Results/Conclusion__ Samples will be run gel on Tuesday, November 15th.

__Reflection__ In this lab, I prepared my samples to be analyzed using qPCR


 * Date: Tuesday, November 8th 2011**
 * Lab 6: Begin personal experiment: Extract RNA from sea urchins**

__Summary__ Purpose: Extract RNA Techniques: Remove tube feet from urchins (20), extract RNA Samples: Taken from Strongylocentrotus droebachiensis

__Materials/Methods__ TISSUE EXTRACTION 1. Place urchins in shallow water 2. Clip tube feet with disection scissors 3. Remove tube feet from solution with tweezers, place each sample in a seperate 1.5ml Tube 4. Label tubes: C1-C6 (controls), P1-P7 (predators), S1-S7 (predator + salinity) 5. Add 500ul Tri-reagent 6. Store samples at -80C

RNA EXTRACTION 1. Homogenize all samples with sterile pestile 2. Add an additional 500ul Tri-reagent 3. Vortex for 15sec 4. Incubate for 5min at room temp, then add 200ul Chloroform under fume hood 5. Vortex for 30sec 6. Incubate for 15s 7. Place into 4C centrifuge at max speed for 15min in order to separate aqueous phase from rest of sample. 8. Transfer clear aqueous phase to new 1.5ml tube. 9. Add 500ul isopropanol to each sample, invert several times 10. Incubate for 10min at room temperature 11. Spin down at max speed for 8min 12. Remove supernatant 13. Add 1ml of 75% ETOH, spin at 7500rpm for 5min 14. Suspend pellet in 100ul of 0.1%DEPC-H2O 15. Nano-Drop all samples (See RNA quantification in lab 2)

Results of Nano-Drop: Reaction ||
 * < Sample ||< ng/ul ||< Amount Needed for1ug of RNA ||< x2ug per
 * < C1 ||< 40.6 ||< 24.6 ||< 49.2 ||
 * < C2 ||< 31.2 ||< 32.1 ||< 64.2 ||
 * < C3 ||< 9.3 ||< 107.5 ||< 215 ||
 * < C4 ||< 22.5 ||< 44.4 ||< 88.8 ||
 * < C5 ||< 17.0 ||< 58.8 ||< 117.6 ||
 * < C6 ||< 22.5 ||< 44.4 ||< 88.8 ||
 * < P1 ||< 61.5 ||< 16.3 ||< 32.6 ||
 * < P2 ||< 17 ||< 58.8 ||< 117.6 ||
 * < P3 ||< 28.2 ||< 35.5 ||< 71 ||
 * < P4 ||< 53.2 ||< x ||< 37.6 ||
 * < P5 ||< 21.3 ||< x ||< 93.9 ||
 * < P6 ||< 28.1 ||< x ||< 71.2 ||
 * < P7 ||< 47.7 ||< x ||< 41.9 ||
 * < S1 ||< 27.6 ||< x ||< 72.5 ||
 * < S2 ||< 60.9 ||< x ||< 32.8 ||
 * < S3 ||< 25.8 ||< x ||< 77.5 ||
 * < S4 ||< 55.4 ||< x ||< 36.1 ||
 * < S5 ||< 47.0 ||< x ||< 42.6 ||
 * < S6 ||< 47.2 ||< x ||< 42.4 ||
 * < S7 ||< 35.5 ||< x ||< 56.3 ||

__Results__ Nanodrop results show a significant amount of sample will be necessary for each reaction due to the small amount of RNA extraction

__Conclusion__ A conclusion will be made after gel is run in a later lab.

__Reflection__ In this lab, I learned how to sample urchin tissue. RNA was extracted, and nano-drop quantification was used to calculate amount of sample to use for each future qPCR reaction.


 * Date: Tuesday, November 1st 2011**
 * Lab 5:** **Agarose gel electrophoresis, protein SDS/PAGE and Western blot**

__Summary__ Purpose: Run amplified PCR products on agarose gel, extract RNA from experiment samples, conduct a western blot for extracted proteins Techniques: Gel electrophoreisis, protein extraction, western blot Samples: taken from C. gigas gill

__Materials/Methods__

GEL ELECTROPHORESIS 1. Using cDNA obtained i lab 4, 25uL of each sample was loaded into aragose gel (including negative controls) 2. 7uL of a 100 bp ladder was loaded into the far left lane 3. Gel was ran at 100V for 1hr 4. Gel was viewed using a UV transilluminator

PROTEIN EXTRACTION AND SDS/PAGE 1. Obtained //C. gigas// gill tissue (0.039g) 2. Transferred tissue to 1.5mL tube labeled with protein, the date, initials 3. Added 500 microliters of CellLytic MT solution to the tube ad homogenize with sterile pestle. 4. Placed sample in a refrigerated microfuge and spun for 10 minutes at max speed. 5. Transfered supernatant to new tube labeled with protein, date, sample, initials, DO 6. Added 15ul of the protein stock to a 1.5mL screw cap tube labeled with protein, initials, date 7. Added 15ul of 2X Reducing Sample Buffer to the 1.5mL screw cap tube 8. Store excess protein at -20C 9. Sample was inverted and boiled for 5min, then immediately cetrofuged. 10. Once the gel was set up in the rig, samples were loaded 11. Ran the gel at 150V for 20min

WESTERN BLOT 1. After protein extraction gel was done, cassette was taken apart and gel was removed 2. Upper right corner was notched in order to maintain proper orientation 3. Filter paper was soaked, membrane and gel in transfer buffer for 15 minutes 4. Blotting sandwich was formed: 1. Annode + 2. filter paper 3. membrane 4. gel facing down 5. filter paper 6. cathode - 4. Blot was allowed to run for 30min at 20V

WASHING (Emma did this) 1. Washed the membrane 2 times for 5 minutes each with 20mL of pure water 2. Placed membrane in plastic box with 10mL of Blocking Solution and incubated overnight on a rotary shaker (1 Rev per Sec) 3. Remove liquid, rinse membrane with 20 mL of water for 5 minutes 4. Incubate membrane in 10mL of Primary Antibody Sol, then decant 5. Rinse with 20mL of Antibody Wash for 5 minutes, repeat three times 6. Incubate in 10mL of Secondary Antibody for 30 minutes, decant 7. Wash the membrane for 5 minutes with 20mL of Antibody wash, repeat three times 8. Rinse with 20 mL of Pure water for 2 minutes, then decant, repeat two times 9. Incubate membrane in 5mL of Chromogenic Substrate until there si a purple band (1-60 min) 10. Dry the membrane in open air on clean filter paper

__Results__ One of our negative controls showed a slight contamination. We did get products from our cDNA, but there were multiple bands. On the western blot, our HSP70 protein did bind to our antibody.

__Conclusion__ Multiple bands on our aragose gel indicate there was probably a small amount of contamination. We had the correct antibody to bind to our heat shock proteins, as we did get product that was expressed.

__Reflection__ In this lab, I learned how run PCR samples and conduct a western blot. Although PCR samples did not turn out perfectly, our western blot did work. Hopefully when we use these procedures for our experiment we will get good, usable results.


 * Date: Tuesday, October 25th 2011**
 * Lab 4: PCR and Conclusion of Personal Experiment**

__Summary__ Purpose: Do a PCR on cDNA with the ordered primers and conclude personal experiments Techniques: Preparing PCR master mix, using a thermocycler Samples: Taken from C. gigas gill

__Materials/Methods__

PCR 1. Spin down primers in a centrifuge 2. Add te buffer to primers (add in uls based on number listed on primer tube) 3. Spin down contents 4. Add 10 ul of the solution to 90 ul di water in a new 1.5 ml tube, which makes 10 micro molar of working solution 5. Make a reaction master in a 1.5 ml tube labeled with MM and initials, adding the following ingredients:


 * || Volume to add per reaction || # of reactions || Total volume to add to MM ||
 * Promega's Go-Taq green master mix || 25 ul || 5 || 125 ul ||
 * Forward Primer || 1 ul || 5 || 5 ul ||
 * Reverse Primer || 1 ul || 5 || 5 ul ||
 * Nucfree H2O || 21 ul || 5 || 105 ul ||
 * ||  ||   || Total Volume: 240 ul ||

6. Pipette 48 ul of master mix into each 0.5 ml PCR tubes labeled with the sample name and initials 7. Add 2 ul of the appropriate template to each tube and mix by pipetting 8. Spin the tubes to collect the liquid at the bottom of the tubes 9. Load the tubes into the thermocycler, making sure the caps are tightly secured 10. Put samples through the following thermocycler profile:


 * 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 ||

11. Store samples at -20C

PERSONAL EXPERIMENT Experiment was concluded. A sufficient amount of tube feet were obtained from each sea urchin, using tweezers and scissors to collect them. One urchin from each tank was dissected, gonad and intestine samples were collected for protein analysis.

Urchin website: http://genefish.wikispaces.com/Green+Sea+Urchin+Predator+Response

__Results__ Hopefully, results will be obtained from PCR. Personal experiment samples can now be analyzed.

__Conclusion__ Results are the same as expected. Next, PCR samples will be run on a gel and analysis of urchin proteins will be carried out.

__Reflection__ In this lab, I learned how to do PCR. This included preparing primers, creating master mix, adding DNA samples to master mix, and running the samples through a thermocycler. I also learned how to collect urchin tube feet and collect urchin gonad and intestine samples. These methods could be used for any kind of DNA analysis or protein collection procedures.


 * Date: Tuesday, October 18th 2011**
 * Lab 3: Reverse transcription and primer design, begin personal experiments**

__Summary__ Purpose: To reverse transcribe RNA to cDNA, design and order primers, and begin our personal experiments Techniques: Using thermocycler, centrifuge, and storing samples at the proper temperature Samples: Taken from C. gigas gill

__Materials/Methods__

REVERSE TRANSCRIPTION 1. Mix stock RNA sample by inverting tube many times 2. Combine the following in a 0.5mL PCR tube (labeled with initials and "cDNA"): 5 uL of total RNA sample, 1 uL oligo dT, 4uL di H2O 3. Incubate the mixture for 5 minutes at 70C, then transfer this mixture to ice 4. Add 5ul of M-MLV 5X reaction buffer, 5ul dNTPs, 1ul of M-MLV RT and 4ul of di H2O 5.Incubate for 60 min at 42C and then heat inactivate at 70C for 3 min 6. Spin down sample in a desk top centrifuge 7. Store at -20C

PRIMER DESIGN To design primers, take the following into consideration: 1. Primers should be 18-30 bases 2. Melting temperatures should be within 2C of each other 3. Avoid primer dimers and hairpins 4. Avoid high G/C stretches, especially at the 3' end 5. G/C clamp at 3' end of primers

PERSONAL EXPERIMENT Experiment will officially begin on Thursday, October 20th when predator is obtained. In the meantime, sea urchins were obtained, and 6, 7 and 7 urchins were placed in 3 tanks respectively. All tanks were filled with bubbled water of a normal salinity. An inch of sand filled about an inch of the bottom of each tank, and small shells of roughly the same size were scattered in an even distribution in half of the tank. Urchins were fead about 2 algal pellets each, and a video camera was set up in order to record the urchin's behavior during the experiment.

Urchin website: http://genefish.wikispaces.com/Green+Sea+Urchin+Predator+Response

__Results__ Hopefully, production of cDNA

__Conclusion__ Results are the same as expected.

__Reflection__ In this lab, I learned about molecular lab techniques as well as experimental design. I learned how to produce cDNA in reverse transcription, how to design primers, and why this is important. I acclimated sea urchins to their tanks, which is a good first step in carrying out the experiment that will ultimately begin on Thursday.


 * Date: Tuesday, October 11th 2011**
 * Lab 2: RNA Extraction and Protein Analysis, Pt. 2**

__Summary__ Purpose: To finish isolating RNA from a whole tissue using TriReagent, then quantify the findings by using a spectrophotometer Techniques: Using TriReagent, using spectrophotometer, recording values, storing samples Samples: Taken from C. gigas gill

__Materials/Methods__

RNA EXTRACTION 1. Incubate tissue sample from lab one at room temperature for 5 minutes 2. Add 200uL of chloroform to sample 3. Vortex sample for 30s, until sample becomes milky 4. Incubate sample at room temperature for 5 minutes. 5. Spin tube in refrigerated microfuge for 15 minutes at max speed. 6. Transfer aqueous phase to a fresh microfuge tube (excluding ALL interphase) 7. Add 500uL isopropanol to RNA (aqueous phase) 8. Mix by inverting tube until the soluttion is uniform 9. Incubate at room temperature for 10 minutes 10. Spin in refrigerated microfuge for 8 minutes at max speed 11. Remove supernatant, leaving white pellet in tube 12. Add 1mL of 75% EtOH to pellet. Vortex to dislodge pellet 13. Spin in refrigerated microfuge for 5 minutes at 7500g 14. Carefully remove only supernatant 15. Spin tube for about 15s to pool residual EtOH 16. Using small pipette tip, remove remaining EtOH 17. Allow pellet to dry at room temperature for no more then 5mins 18. Add 100uL of 0.1%DEPC-H2O to tube, re-suspend by pipetting up and down until pellet is dissolved 19. Incubate tube at 55C for 5mins to help solubilize RNA 20. Remove tube from heat, flick to mix and place this stock RNA sample on ice. 21. Use Nanodrop spectrophotometer to quantify RNA yield

RNA QUANTIFICATION 1. Pipette 2uL of 0.1%DEPC-H2O onto the Nanodrop pedestal and lower arm 2. Zero the instrument by clicking blank 3. Pipette 2uL of RNA sample onto pedestal and lower arm 4. Click Measure. Record RNA concentration (ng/uL), A260/280 ratio and A260/230 ratio. 5. Raise the arm and wipe off sample with a KimWipe 6. Label stock RNA sample with "RNA", source organism/tissue, initials, today's date and the concentration in ug/uL 7. Store samples at -80C.

__Results__ ng/ul = 292.3 A260/280 ratio = 1.85 A260/230 ratio = 1.94

__Conclusion__ RNA extraction results showed a good ratio of 1.85.

__Reflection__ The purpose of this lab was to understand how to extract RNA and use a Nanodrop specophotometer. Lab procedures measure RNA extraction quality. These methods could be used for any study where the extraction of RNA is necessary.


 * Date: Tuesday, October 4th 2011**
 * Lab 1: RNA Extraction and Protein Analysis, Pt. 1**

__Summary__ Purpose: To Isolate RNA and protein from a whole tissue, and to determine the concentration of total protein in the sample. Techniques: Use TriReagent to isolate RNA, and CelLytic MT to isolate protein. Use the Bradford protein assay to determine the concentration of total protein in the tissue. Samples: Taken from C. gigas gill (RNA extraction) and digestive gland (protein extraction). user:emmats

__Materials/Methods__ 1. Ice sample until ready for homogenization (C. gigas gill was used) 2. Add 500uL TriReagent to tissue 3. Homogenize and vortex sample - how did you homogenize? user:emmats 4. Add 500uL more TriReagent to tissue 5. Vortex for 15 seconds 6. Store sample at -80 degrees C

1. Dilute protein sample 1:2, so there is a total of 30uL in the tube 2. Make a blank with 30uL DI water 3. Add 1.5mL Bradford reagent to each tube, invert them and incubate at room temperature for 10 minutes 4. Mix both tubes and transfer the contents to cuvettes 5. Zero the spectrophotometer with the blank and measure the absorbance at 595nm. 6. Mix, take another measurement 7. Average the values and calculate the protein concentration using the bradford protein assay

__Results__ 1. RNA sample from C. gigus gill, 0.02g

2. Protein sample from C. gigus digestive gland, 0.053g

1st 595nm absorbance: 0.261 2nd 595nm absorbance: 0.261 Average absorbency: 0.261

Applied to bradford protein assay: y=mx+b x=0.261 m=1013.9 b=0 Multiplied by 2 because of the 1/2 ratio y=0.261*1013.9*2 y=1013.9*0.523 y=530.270

__Conclusion__ The results of the RNA extraction have yet to be determined, but the results of the protein extraction lie within the expected range and are of a fairly intermediate concentration. Next, I am going to finish the RNA extraction and calculate the RNA yield.

__Reflection__ The purpose of this lab was to get familiar with the techniques of RNA and protein extraction, as well as to use a spectrophotometer and apply the protein measurements taken to the bradford protein assay. Procedures are used to measure the protein concentration. These methods would be used for analyzing RNA sequences, and proteins produced. This study could look at an animals RNA sequence, and relate it to proteins produced through translation. Nothing about the procedure was unclear, however I wish that there was more specifics on how the bradford protein assay works.