Mikhail's+Notebook


 * Lab # 10: qPCR of Olympia Oyster cDNA (from gill tissue)**
 * 11/28/12-11/30/12**

The purpose of this lab was to prepare the cDNA created from Olympia Oyster mRNA during last week’s lab, for quantitative polymerase chain reaction (qPCR). A master mix was created, enough for the 30 cDNA samples, 2 blanks, and 2 extra samples to account for the possibility of pipetting error. cDNA was added to the master mix and ran through multiple PCR cycles.
 * Summary of Lab:**

PCR Plates (white); optically clear caps, 1.5 ml microfuge tubes (RNAse free), Nuclease Free water, filter tips, Opticon thermal cycler, kim wipes, 2x SensiMix, SyBR 25x dye, microfuge tube racks, ice buckets, timers, cDNA samples (student provided), Lab coat, Safety glasses, GlovesMethods:
 * Materials**:

Create MM including the following: (note: 34 X the following components) For a 25μl reaction volume: ComponentVolume Final Conc. 2x SensiMix12.5µL 1x SyBR 25x dye1µL 2µM upstream primer, 10μM1.25μl 2.5μM downstream primer, 10μM1.25μl 2.5μM PCR Water 7uL NA
 * Methods**:

1. Obtain 5 white PCR plates (should contain 8 wells each) and add 23 µL of MM to each. Should have rows of 10, so need to break two of the wells in half. 2. Add 2ul of cDNA template to 30 wells, leaving two into which you need to add 2ul of PCR H2O. 3. Cover wells tightly with caps, and clean off with kimwipes, leaving the lids clean. 4. TA will then Load plate into PCR and run under the following conditions: PCR conditions:1. 95°C for 10 minutes2. 95°C for 15s3. 55 °C for 15 s4. 72°C for 30 s (+ plate read) 5. Return to step 2 39 more times6. 95°C for 10s7. Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

Results will be available following the PCR process in several days. The next step will be to analyze the results in excel and determine whether or not gradual temperature stress causes an increase in gene expression. user:macgavery need to include results adn interpret
 * Results/ conclusion:**

The purpose of this lab was to use our knowledge about PCR. The procedures in this lab were used to measure gene expression in tissue samples of Olympic oyster gills. These methods are used for studies looking at gene expression in organisms and comparing gene expressions prior to, during and after stresses are applied. Nothing was unclear about the procedures used and there was plenty of information to understand the lab and its overall purpose.
 * Reflection:**


 * Lab #8 & 9: RNA Isolation of Olympia Oyster Gill tissue samples**
 * 11/12/12 - 11/20/12**


 * Summary of the Lab:**

This lab was a continuation of the research project for the class, following the experimentaltrails and collecting of tissue and measuring of oysters a couple weeks ago. The focus of this week’s lab was to Isolate and extract RNA from the tissue samples collected, to be used in cDNA synthesis during next week’s lab.


 * Materials:**

micropipettes (1-1000 μL), sterile filter pipette tips (1-1000 μL), 1.5 mL microcentrifuge tubes, sterile disposable pestles, vortex, TriReagent, microcentrifuge tube rack, lab coats, safety glasses, gloves, lab pen, timers, 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.


 * Methods:**


 * NOTE: perform each step for each sample # before moving on to next step (12 x total)**

Sample #’s 58-60, 65-68, 71-75 (12 total) My samples:

Treatment (Olympia oyster gill) Sample #’s 14°C Control 58-60 (3 total) 35°C (ambient) 65-68 (4 total) Gradual increase from 14°C to 35°C 71-75 (5 total)

1. Extract 50-100mg of tissue from sample and place into 1.5ml snap cap tube labeled with yourinitials, date, and sample # with a lab pen. 2. After sample is inside closed and labeled snap cap tube, place tube on ice. 3. Add 500ul of TriReagent to tube and place back on ice. 4. Use a disposable pestle to homogenize tissue (tissue should break apart and dissolve). It tissuedoes not homogenize well, you could briefly vortex sample. 5. Once homogenized completely, add another 500ul of TriReagent to tube, close tightly andvortex for 15 sec. 6. Under the fume, add 200uL of chloroform to tube. Note: Pipetting needs to be done carefullyand quickly, as chloroform is highly unstable. Chloroform container should be closed at all timeswhile no directly pipetting from container.

7. Using one of several vortex machines, mix tube for 30 seconds. After this step is completed, thechloroform containing sample should have a milky appearance.

8. Incubate at room temperature once again for 5 minutes.

9. Spin tube at max speed for 15 minutes in refrigerated microfuge, located near the front of the lab.

10. Carefully remove tube, as to not mix content inside tube. Note: You should notice three layers;the aqueous phase (top, clear portion), an interphase (middle portion containing cell debris) andthe organic layer (bottom portion).

11. Transfer most of the aqueous phase (without disturbing the interphase) to a new 1.5 mLmicrocentrifuge tube.

12. Close and dispose remainder of old tube into appropriate bins (including microcentrifuge tube) atend of lab.

13. To the new tube, Add 500uL of isopropanol and close tightly.

14. Mix this solution by continuously flipping upside-down until solution is uniform in appearance (nolonger lumpy or viscous), and store at room temperature for 10 minutes

15. Place tub into refrigerated microfuge and spin at max speed for 8 minutes. Note: while inmicrofuge, tube hinge should be pointing up and away from center of microfuge, as to allow alltubes to be uniform.

16. Remove supernatant (The bottom of tube should contain a “white pellet” of RNA and salts). 17. It should now contain only a pellet. Addc1 mL of 75% EtOH to sample B and vortex once again,until pellet is free from tube walls. Note: it’s OK if pellet is not dislodged. 18. Use refrigerated microfuge to spin tube at 7500g for 5 minutes. 19. Remove supernatant, leaving only the pellet behind once again, and spin tube briefly to extractany remaining EtOH. 20. Using small pipet, remove the remainder of EtOH in tube. 21. Open tube for up to 5 minutes to allow pellet to dry at room temperature. 22. Add 100uL of 0.1% DEPC-H2O to tube and pipet up and down to dissolve pellet completely. 23. Store sample B at 55°C for 5 minutes. This will help solubilize RNA. 24. Remove tube and flick it to mix. 25. Place sample into -80°C freezer to be used for cDNA synthesis in the following lab.

No results yet during this lab; however, once qPCR is complete, we will be running samples ongels and quantitating the translation of genes of interest.
 * Results:**

Because I’ve now had practice performing RNA isolation, this portion of the project has ransmoothly and with only very minor errors. The next step will be to create complementary DNA (cDNA)strands from the extracted RNA and to use that cDNA in qPCR, after which we will finally see the resultsof our experiment al trails.
 * Conclusion:**

The purpose of this lab was to perform one of the first steps in looking at changes in geneexpression, and that is to isolate RNA from tissue samples, and in our case, from oysters. Theprocedures in this lab were used to measure effects of gradual vs. acute temperature increase onOlympia oysters. These methods may be used for any study aiming to understand how environmentalfactors such as temperature change effects the fitness of a variety of organisms. Nothing was unclearabout this lab, as it is now the second time I have performed these procedures, and there was plenty ofinformation to draw from.
 * Reflection:**

Lab #6: Conventional PCR (from Lab #5), qPCR & Protein isolation 10/30/12

The purpose of this lab was to preform cPCR on oyster gill tissue samples obtained during lab #5 and to perform qPCR on the cDNA isolated during a previous lab. These tasks were performed w/ the help of “master mixes”, which included our primers, tissue samples and other reagents which would assist in PCR.
 * Summary of the lab: **

Materials: Micropipettes (1-1000 μl), Sterile filter pipette tips (1-1000 μl), Tip waste jar, PCR tubes (0.5 ml; thin walled), 1.5 ml microcentrifuge tubes (RNAse free), cDNA (student provided), dNTPs, 2x GoTaq Green Master Mix, Primers, Nuclease Free water, thermal cycler, Kimwipes, microfuge tube racks, PCR tube racks, ice buckets, Lab coat, Safety glasses, Gloves
 * cPCR **

Methods: Create a Mastermix containing the following reagents in a 1.5 ml microcentrifuge tube and label it as “MM”, include initials.
 * Reagents ||  ||
 * 5x GoTaq Green buffer || 50 µL ||
 * 10mM dNTP mix || 5 µL ||
 * 10 µM forward primer || 5 µL ||
 * 10 µM reverse primer || 5 µL ||
 * GoTaq polymerase || 1.25 µL ||
 * nuclease-free water || 11.75 ||


 * 1) Extract 48 ul of MM into 4 different .5 ml PCR tubes, 2 labeled w/ “negative control” and your initials, and two different tissue samples w/ sample names and your initials.
 * 2) Close tube caps and spin to move all liquid to bottom of tubes.
 * 3) Samples will be put through thermal cycling profile, which includes the following steps, after which they will be stored at -20°C for later use.


 * 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  ||

Results: The results of the cPCR will only be known after the thermal cycling is complete and the DNA is ran on gels.

Conclusion: The next steps will be to run samples on gels and see if the target genes are present.

Materials: PCR Plates (white); 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, cDNA samples (student provided), Lab coat, Safety glasses, Gloves
 * qPCR **

Methods:

Create another MM including the following // For a 25μl reaction volu // me:
 * 5 X the following components **
 * **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) Obtain white PCR plates (should conatin 8 wells) and add 25 µL of MM to each.
 * 2) Using your cDNA from a previous lab, add 2ul of cDNA template to several wells, leaving atleast two into which you need to add 2ul of PCR H2O.
 * 3) Cover wells tightly with caps, and clean off with kimwipes, leaving the lids clean.
 * 4) TA will then Load plate into PCR and run under the following conditions:

PCR conditions:1. 95°C for 10 minutes2. 95°C for 15s3. 55 °C for 15 s4. 72°C for 30 s (+ plate read)5. Return to step 2 39 more times6. 95°C for 10s7. Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

Results: Results were not obtained during this lab, but will be known following PCR, in a later lab. Conclusion: While no particular results were expected, after the PCR is completed, we will be able to possibly detect and quantify targeted DNA molecules (genes).

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, gloves, Kim wipes, lab pens, safety glasses, CelLytic MT Cell Lysis Reagent (with Protease Inhibitor Cocktail added), Coomassie Protein Assay Reagent, DI water, Lab coat, Safety glasses, Gloves
 * Protein isolation **

Methods: Protein extraction protocol NOTE: //Each step is done twice, as there are two tissue samples//
 * 1) Extract 25mg of tissue sample and place into 1.5 ml microfuge tube. (record tissue weight)
 * 2) Label cap with initials and todays date.
 * 3) Extract 500 ul of CellLytic MT solution and pipet into tube.
 * 4) Using sterile, disposable pestle, homogenize tissue. Close tube and invert several times to mix.
 * 5) With other people, mix samples in refrigerated microfuge for 10 minutes at max speed.
 * 6) Label a new 1.5 ml tube with the word “Protein”, your initials, date, and the organisms name and tissue type (e.g. gill).
 * 7) Once sample is done spinning, transfer the supernatant, or liquid layer to your new tube and store on ice.

Methods: Protein quantification protocol NOTE: //Each step is done twice, as there are two tissue samples// // Do steps 7 & 8 twice, for blank and protein sample. //
 * 1) Label 2 ml screw cap tube w/ initials, the date the word “Protein” and “BA”.
 * 2) Pipet 15 uL of protein sample and 15 uL of DI H2O into your tube and mix by pipetting.
 * 3) Obtain a second 2 ml screw cap tube and pipette 30 uL of DI water (this is for your blank and only needs to be done once per group).
 * 4) Add 1.5 mL of Bradford reagent to both tubes.
 * 5) Close tubes tightly, invert several times to mix and keep at room temperature for 10 minutes.
 * 6) Transfer 1000 ul of both samples to plastic, disposable cuvettes.
 * 7) Calibrate the spectrophotometer using the blank tube. (remember to wipe the cuvettes on your tubes first with a Kimwipe to remove any finger prints)
 * 8) Measure your absorbance at 595nm and record value.
 * 1) Using the standard curve below, back-calculate protein concentration.
 * 2) Return remainder of protein sample to TA to be stored @ -20C.

Bottom of Form

Results:

G3 absorbance @ 595 nm = 0.299; Protein concentration = 303.16 ug/mL

G2 absorbance @ 595 nm = 0.325; Protein concentration = 329.52 ug/mL

Conclusion: The protein concentration in gill sample # 2 was 26.36 ug/mL lower than that of gill sample # 3. Both of these oysters were exposed to a common stress. Differences in gene expression could be due to oyster size, or other factors associated with different results such as standard error, or calculating error. The next step will be to compare these results with that of other students who examined oysters under other stresses.

The purpose of this lab was to use our new knowledge about PCR and extraction of proteins on our oyster samples. The procedures in this lab were used to measure gene expression in tissue samples of Olympic oyster gills. These methods are used for studies looking at gene expression in organisms and comparing gene expressions prior to, during and after stresses are applied. Nothing was unclear about the procedures used and there was plenty of information to understand the lab and its overall purpose. --
 * Reflection: **

Lab #5: tissue dissection, primer reconstitution, end-point PCR 10/23/12 Olympia oysters from all sections of our experiment were measured, shucked and dissected upon completion of their treatment. Tissue samples from these oysters were placed into microcentrifuge tubes and into a freezer to be analyzed in next week’s lab. Primers (in a dehydrated state) were re-hydrated and stored in freezer for next week’s lab.
 * Summary of the Lab: **

Shuckers Measurers Experiment Oysters Microcentrifuge tubes Ice buckets Lab coat Gloves Tip waste jar Primers Nuclease Free water Microfuge tube racks Tip waste jar Sterile filter pipette tips Micropipettes Lab pen Razor blsades Tweezers Notebook Pen Marker
 * Materials: **

All of the oysters survived the various treatments as they were all tightly closed prior to shucking, however we did discover a jingle shell amongst our oysters which did not survive the temperature change from 14C to 35C. While this week’s lab was a lot of manual labor in shucking and dissecting the oysters, the fallowing labs will hopefully reveal interesting results pertaining to our treatments.
 * Methods: Tissue Dissection **
 * 1) Prepared Olympia oyster experimental groups were extracted from saltwater one at a time and length + width were measured. All measurements were recorded.
 * 2) Oysters were placed into trays and shucked by hand.
 * 3) Shucked oysters were placed back into try and gill + mantle tissue was extracted.
 * 4) Tissue was placed into separate microfuge tubes and labeled with specimen number, oyster type, tissue and treatment type.
 * 5) Labeled Microfuge tubes were placed into ice buckets.
 * 6) Once all oysters were shucked, microfuge tubes were placed into freezer for RNA extraction during next week’s lab.
 * 7) Oyster “left overs” were disposed into waste disposal container and work area was cleaned.
 * Results/ conclusion: **

I think that the purpose of this lab was for us to become familiar working with living organisms and teaching us various methods of sampling them. We did several sampling techniques, including: recording oyster shell length and width, and removing gill and mantle tissue. The methods performed in this lab might be used in studies trying to answer the questions of how organisms respond to environmental change on a molecular and RNA level. While nothing was unclear about this lab, it would have been helpful if the lab has hints on dissecting techniques as that seemed to be quite frustrating at some points for the people doing the dissecting. -- 10/16/12 Lab # 4: Reverse transcription and primer design & Prep for Experiment RNA was reverse transcribed into complementary DNA (cDNA). Also, we learned about oyster anatomy and shucking techniques. A Mock-up was done in preparation for the experiments beginning the following weak in lab, and primers were designed using the __NCBI Primer__ program.
 * Reflection ** :
 * Summary of Lab**

Micropipettes (1-1000 μl) Sterile filter pipette tips (1-1000 μl) Tip waste jar PCR tubes (0.5 ml; thin walled) RNA samples (student provided) M-MLV reverse transcriptase M-MLV 5X reaction buffer Oligo dT dNTPs Nuclease Free water thermal cycler microfuge tube racks PCR tube racks ice buckets Kimwipes Lab coat Safety glasses Gloves
 * Materials: Reverse transcription**

Oyster shuckers protective gloves 10% bleach and sand in a beaker Dilute ethanol in a beaker Scalpel and blades Forceps
 * Materials: Sterile Dissection ** (not done in this week’s lab)

Containers Heating elements Water baths Airstones Air pumps
 * Materials: Experiment Mock-up **


 * Methods: Reverse transcription**
 * 1) Recover stock RNA from last week’s lab (//Sample B//) and mix content by inverting tube 4 several times.
 * 2) Combine 1 ul oligo dT, 4 ul nuclease free H20 and 5ul of //Sample B//.
 * 3) Let new solution, //Sample C// sit for 5 minutes at 70C on thermocycler.
 * 4) Once complete, emediatly transfer //Sample C// to ice bucket until you could centrifuge the solution for several seconds.
 * 5) Add 5 ul M-MLV 5X Reaction Buffer, 5 ul dNTPs, 1 ul M-MLV RT and 4ul of nuclease free H2O to //Sample C//.
 * 6) Rest mixture at 42C for 60 minutes.
 * 7) Heat for 3 min at 70C on thermocycler.
 * 8) Mix sample briefly on dest top centrifuge and store //Sample C// at -20C or in ice.


 * Methods: Primer Design**
 * 1) Using the internet, find a gene interesting to you, within your species of interest.
 * 2) Enter scientific name of species and gene of interest into the __NCBI Primer__ tool search bar on the NCBI home page ([]).
 * 3) Select an RNA sequence from the results and design primer (to be ordered for following labs).
 * Methods: Sterile Dissection** (not done in this week’s lab)

Working in teams, decide how different parts of the experiment should be set up. You may use the materials listed above to create a mock-up of the experiment you will be doing next week.
 * Methods : Experiment Mock-up**

No results are obtained from this lab.
 * Results/ Conclusion:**

There were a few purposes for completing this lab. During this lab we learned how to synthesized cDNA from RNA using reverse transcription. We also “mastered” the technique of shucking and eating oysters, as well as learning a little about the anatomy of oyster. Finally, we set up out experiments for next week and discussed how they would be performed. Some of the procedures in this lab are used to make double stranded DNA from RNA, which could then be replicated via DNA Replication. This results in a more stable code which could be analyzed to measure gene expression. These methods may be used in studies looking at physiological change in an organism experiencing environmental stress, such as the oysters we will be looking at. The procedures in this lab were very clear and everything made sense as the lab moved along. I wish there was a little more information on the steps required to set up and design RNA primers using the NCBI website.
 * Reflection:**

10/09/12 Lab # 3: RNA Isolation, Part 2 This lab was a continuation of lab 2, where we began extracting RNA from oyster gill tissue. The homogenized tissue samples were repeatedly incubated and Spun in a refrigerated microfuge as chemicals were added to isolate and extract RNA from cell tissue. When a pure RNA sample was achieved, RNA yield was able to be quantitated using Nanodrop spectrophotometry. RNA Extraction RNA Quantification Note: the first 2 steps only need to be done once per class. RNA Concentrations (using the Beer-Lambert law): Absorbance: 26.047 A260/280 ratios: 1.98 A260/230 ratios: 1.16 RNA concentration: 1213.5 ng/uL While my A260/280 ratios are within the normal range of 1.5-2.0, indicating that at this absorbance, my RNA is fairly clean. However, my A260/230 ratios are lower than the suggested range of 1.5-2.0. This might result from substances other than RNA in my sample, such as Ethanol or salt. These results, along with an RNA concentration of 1213.5 ng/uL and an absorbance of 26.047 are within the range of what I expected. There were many steps in the procedures for something to go wrong or be miscalculated, and as this is my first advanced molecularbased lab, It will take practice to perfect the method. The purpose of this lab is to introduce students to molecular techniques and methods which will be helpful later this quarter and in future labs. The procedures in this lab are meant to isolate RNA from cells and other molecules; when RNA is extracted and a pure sample is achieved, it is possible to quantify the sample’s RNA concentrations using Nanospectrometry. These methods could be used to study responses by organisms to environmental stresses. All of the procedures used in this lab had a clear purpose to me. More background information into how the different chemicals we add to our samples would help me visualize what exactly it is that we are doing to the RNA, step by step.
 * Summary of the Lab**
 * Materials**
 * micropipettes (1-1000 μL)
 * sterile filter pipette tips (1-1000 μL)
 * 1.5 mL microcentrifuge tubes
 * microcentrifuge tube rack
 * lab coats
 * safety glasses
 * gloves
 * lab pen
 * timers
 * 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
 * Methods**
 * 1) Remove homogenized tissue sample (//sample A//) created during lab 2 from storage at -80*C and allow it to rest at room temperature for 5 minutes.
 * 2) Under the fume, add 200uL of chloroform to //sample A//. Note: Pipetting needs to be done carefully and quickly, as chloroform is highly unstable. Chloroform container should be closed at all times while no directly pipetting from container.
 * 3) Using one of several vortex machines, mix //sample A// for 30 seconds. After this step is completed, the chloroform containing sample should have a milky appearance.
 * 4) Incubate at room temperature once again for 5 minutes.
 * 5) Spin //sample A// at max speed for 15 minutes in refrigerated microfuge, located near the front of the lab.
 * 6) Carefully remove //sample A//, as to not mix content inside tube. Note: You should notice three layers; the aqueous phase (top, clear portion), an interphase (middle portion containing cell debris) and the organic layer (bottom portion).
 * 7) Transfer most of the aqueous phase (without disturbing the interphase) to a new 1.5 mL microcentrifuge tube, (//sample B//).
 * 8) Close and dispose remainder of //sample A// into appropriate bins (including microcentrifuge tube) at end of lab.
 * 9) To //sample B//, Add 500uL of isopropanol and close tube.
 * 10) Mix //sample B// by continuously flipping upside-down until solution is uniform in appearance (no longer lumpy or viscous), and store at room temperature for 10 minutes
 * 11) Place //sample B// into refrigerated microfuge and spin at max speed for 8 minutes. Note: while in microfuge, tube hinge should be pointing up and away from center of microfuge, as to allow all tubes to be uniform.
 * 12) Remove supernatant (The bottom of //sample B// should contain a “white pellet” of RNA and salts).
 * 13) //Sample B// should now contain only a pellet. Addc1 mL of 75% EtOH to //sample B// and vortex once again, until pellet is free from tube walls. Note: it’s OK if pellet is not dislodged.
 * 14) Use refrigerated microfuge to spin //sample B// at 7500g for 5 minutes.
 * 15) Remove supernatant, leaving only the pellet behind once again, and spin //sample B// briefly to extract any remaining EtOH.
 * 16) Using small pipet, remove the remainder of EtOH in //sample B//.
 * 17) Open tube for up to 5 minutes to allow pellet to dry at room temperature.
 * 18) Add 100uL of 0.1% DEPC-H2O to //sample B// and pipet up and down to dissolve pellet completely.
 * 19) Store //sample B// at 55*C for 5 minutes. This will help solubilize RNA.
 * 20) Remove //sample B// and flick tube to mix.
 * 21) You now have your stock RNA sample which should be kept on ice (at ALL TIMES) and will be used in the RNA quantification portion of this lab.
 * 1) Start calibrating Nanospectrometer by pipetting 2uL of 0.1% DEPC-H2O on the Nanodrop pedestal and lowering the arm.
 * 2) On the computer screen, select “Blank” setting the default concentration to zero.
 * 1) Clean off the Nanodrop pedestal using a Kimwipe and proceed to pipette 2uL of RNA sample (//sample B//) onto the Nanodrop pedestal, and lower the arm.
 * 2) Click “Measure” for the Nanospectrometer to calculate the RNA concentration (ng/uL), the A260/280 and the A260/230 ratios of //sample B//. Note: the Beer-Lambert law is used by the Nanodrop to calculate your RNA concentrations.
 * 3) Wipe off your sample from the Nanodrop pedestal with a KimWipe and “CLEARLY” label //sample B// with: The word “RNA” 2, source organism/tissue (e.g. gill tissue), your initials, todays date and the RNA concentration in ug/uL.
 * 4) Return your sample to the TA to be stored at -80*C.
 * Results **
 * Conclusion **
 * Reflection **

10/02/12 Lab 2: DNA isolation; initiate RNA isolation DNA and RNA were extracted from the gill tissue of Olympia Oysters using TriReagent to separate RNA from various cells and DNazol to remove DNA from other cells in the tissue. After isolating RNA and DNA from cells, the samples could then be quantified using spectrophotometry and their concentration could be determined. The purpose of this lab was to gain a better knowledge of how essential information about a particular organism could be extracted on a molecular scale. The methods learned in lab could be applied to any organism and will be useful in future labs and experiments.
 * Summary of the Lab:**


 * Lab Objectives ** //By the end of lab you should have completed the following: //


 * Select tissue for DNA, RNA and protein extraction
 * Start RNA extractions (add Tri-Reagent to samples, homogenize, and then re-freeze for next week)
 * Isolate and quantify DNA from tissue.

RNA Extraction Part 1

 * Supplies and Reagents **


 * micropipettes (1-1000uL)
 * sterile filter pipette tips (1-1000uL)
 * sterile (RNase free) 1.5mL microcentrifuge tubes
 * sterile disposable pestles
 * vortex
 * ice buckets
 * gloves
 * lab pens
 * safety glasses
 * <span style="background-color: #ffffff; color: #000000; font-family: Arial,Helvetica,sans-serif;">TriReagent


 * You will isolate RNA from whole tissue using [|TriReagent]. TriReagent allows for separation of RNA from other cellular components, including DNA. There are three primary components of TriReagent that allow this to happen. The first is guanidine isothiocyanate which is a potent protein denaturant, the second is phenol, and the third is pH.
 * Guanidine isothiocyanate denatures proteins, such as the highly abundant histones that coat DNA. Even more importantly, RNases are denatured. This denaturing action allows for better access of phenol (an organic solvent) to cellular proteins and improves its ability to keep the proteins insoluble. The pH of TriReagent is acidic. The low pH keeps DNA out of solution while RNA remains soluble.
 * After homogenizing/lysing your tissue in TriReagent, chloroform (another organic solvent) will be added to your sample to allow for separation of the phenol and insoluble cellular components (DNA, proteins) from soluble cellular components (RNA). This will result in three distinct layers: the organic phase (the bottom portion), the interphase (layer of cell debris) and the aqueous phase (the top portion). The aqueous phase (the RNA) can then be easily isolated.
 * The RNA can be precipitated and washed to remove residual phenol and salt carryover. Then the RNA can be resuspended in a suitable solution and quantitated.
 * RNA is quantitated using a spectrophotometer and measuring the absorbance of your RNA sample at 260nm (A260). The concentration of your sample is calculated with the following equation:[RNA] = 40ug/mL x A260 x Dilution Factor
 * In addition to the A260, absorbance at 230nm and 280nm should be taken. The ratio of A260 to each of these absorbances can be used to assess the purity of your RNA. Various substances will absorb at 230nm, which will indicate carryover of phenol, ethanol or high salt in your sample. Proteins generally absorb light at 280nm. For clean RNA, A260/A280 should range between 1.8-2.0. The A260/A230 should range between 1.5-2.0 for clean RNA.


 * RNA ISOLATION PROTOCOL **


 * 1) Label the snap cap tube containing your tissue sample with your initials and the date using a lab marker. Keep the sample stored on ice until you are ready for homogenization.
 * 2) Add 500uL of TriReagent to the 1.5mL snap cap tube containing your tissue. Store on ice.
 * 3) Carefully homogenize the tissue using a disposable pestle. If the tissue is difficult to homogenize, carefully close the tube tightly and briefly vortex the sample.
 * 4) After the sample is completely homogenized, add an additional 500uL of TriReagent to the tube and close the tube tightly.
 * 5) Vortex vigorously for 15s.
 * 6) //Stop here for Lab 1 and give your **labeled** homogenized tissue sample to the TA for storage at -80ºC. You will be finishing your RNA extraction in lab next week.//

__** DNA Isolation (DNazol) **__

 * Supplies and Reagents **


 * micropipettes (1-1000 µL)
 * sterile filter pipette tips (1-1000 µL)
 * 1.5 mL microfuge tubes
 * microcentrifuge tube rack
 * microcentrifuge (room temperature)
 * razor blades
 * vortexes
 * DNazol
 * 100% ethanol
 * 75% ethanol
 * 0.1% DEPC water
 * kim wipes
 * Nanodrop


 * Procedure Background **


 * DNazol uses a guanadine-detergent lysing solution to hydrolyze RNA and selectively precipitate DNA from the cell. During the extraction, the cells in the sample are lysed and DNA is precipitated using ethanol. DNA is then solubilized using water or NaOH. The genomic DNA recovery should be between 70 and 100% using this protocol.


 * DNazol is an irritant, so please be careful and wear protective gear when using it.
 * Centrifuging in step 3 removes insoluble tissue fragments, partially hydrolyzed RNA, and excess polysaccharides. This step is necessary to get higher quality DNA.
 * If you have trouble removing your DNA precipitate in step 8, you can centrifuge it at 5,000 x g for 5 minutes (room temperature) so that the DNA will form a pellet.
 * We will "spec" our DNA after extraction. Purified DNA should have a A260/A280 ratio of 1.7-1.9, indicating good quality DNA.


 * DNazol Extraction Protocol (Adapted from MRC manual) **


 * 1) Using a sterile pestle, homogenize your tissue sample in 0.5 mL of DNazol in a 1.5 mL sterile microfuge tube. After the tissue is homogenized, add 0.5 mL more of DNazol and mix well.
 * 2) Let your sample incubate for 5 minutes at room temperature.
 * 3) Spin your sample at 10,000 x g (room temp) for 10 minutes.
 * 4) Transfer your supernatant to a new, labeled tube.
 * 5) Add 0.5 mL of 100 % ethanol to your sample.
 * 6) Mix your sample by inverting your tube 5-8 times.
 * 7) Store your sample at room temperature for 1 minute.
 * 8) Your DNA should form a cloudy precipitate. Remove the DNA and put in a new tube using your pipette.
 * 9) Let your sample sit at room temp for 1 minute and remove the rest of the lysate (liquid that is not DNA).
 * 10) Wash your DNA with 1 mL of 75% ethanol: Pipette the ethanol into your DNA tube, invert 6 times, and let sit for 1 minute. Remove the ethanol from the tube and repeat.
 * 11) If there is ethanol left at the bottom of your tube after the second wash, remove with a small pipette.
 * 12) Add 300 µL of 0.1% DEPC water to your DNA and pipette up and down multiple times to dissolve.
 * 13) Bring your DNA sample up to the Nanodrop to quantify.


 * DNA Quantification **


 * 1) Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm.
 * 2) Select "dsDNA" from the pulldown menu
 * 3) Click "Blank", to zero the instrument. NOTE: steps 1 and 2 only need to be done once for the whole class.
 * 4) Pipette 2µL of your DNA sample onto the Nanodrop pedestal and lower the arm
 * 5) Click "Measure". Record your DNA concentration (ng/µL), A260/280 ratio and A260/230 ratio. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate DNA concentration for you.
 * 6) Raise the arm and wipe off you sample with a KimWipe
 * 7) Clearly label your stock DNA sample with the word "DNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
 * 8) Store sample at -20ºC.