Selina+Cho


 * Lab One: RNA Extraction and Protein Analysis Part One**


 * Date**: October 4, 2011

The process of RNA isolation and extraction from a //Crassostrea gigas// gill tissue was initiated using TriReagent. In addition, protein from a //Crassostrea gigas// digestive gland tissue was isolated using CelLytic MT and protein concentration was determined using the Bradford Protein Assay.
 * Summary of the Lab:**

__RNA Isolation and Extraction__
 * Materials and Methods:**
 * //Crassostrea gigas// tissue samples
 * 1000µL and 20µL pipettes
 * 1000µL and 20µL sterile pipette tips
 * sharpie - sharpie
 * sterile disposable pestles
 * vortex - Ice buckets
 * ice buckets
 * RNase free 1.5mL microcentrifuge tubes
 * safety glasses
 * lab coats
 * TriReagent
 * tweezers
 * 10% bleach solution

__Protein Extraction and Analysis__
 * //Crassostrea gigas// tissue samples
 * 1000µL and 20µL pipettes
 * 1000µL and 20µL sterile pipette tips
 * sharpie
 * sterile disposable pestles
 * Ice buckets
 * RNase free 1.5mL microcentrifuge tubes
 * spectrophotometer
 * cuvettes
 * kim wipes
 * safety glasses
 * lab coats
 * Sterile 2mL screw cap microcentrifuge tubes
 * microcentrifuge in fridge
 * CelLytic MT Lysis Reagent with Protease Inhibitor cocktail added
 * Coomassie protein Assay Reagent= Bradford Assay
 * DI water

__RNA Isolation and Extraction__ 1. 0.013g of //Crassostrea gigas// gill tissue is transferred to a RNase free 1.5mL microcentrifuge tube using sterilized tweezers and the tube is labeled with initials. the contents, and the date - would be helpful to write down exactly what the label is user:emmats 2. 500µL of TriReagent is added to the 1.5mL tube and a disposable pestle is used for homogenization 3. the tube is briefly vortexed to allow for better homogenization 4. when complete homogenization is determined, add another 500µL of TriReaget, close cap, and vortex for 15 seconds 5. After vortexing, give the tube for TA - where was it stored/at what temperature? user:emmats

Observations/Deviations: point of the disposable pestle was to wide so it did not go all the way into the 1.5mL tube causing some tissue pieces to not be completely homogenized

__Protein Extraction and Analysis__ 1. 0.019g of //Crassostrea gigas// digestive gland tissue is transferred to a 1.5mL tube with sterilized tweezers 2. the tube is labeled with initials, the contents, and the date 3. 500µL of CelLytic MT solution is added to the tube and a disposable pestle is used for homogenization 4. close the tube, invert several times, and place it into a refrigerated microfuge for 10 minutes at max speed 5. Label a new tube as "protein sample" and when 10 minutes has passed, transfer only the clearish liquid from the microfuged tube 6. Label a new 2mL screw cap tube as "Protein BA", initials, and the date 7. pipette 15µL of your "protein sample" and 15µL of DI water into the 2mL tube which equals to a 1:2 sample 8. In a second 2mL tube, add 30µL of DI water serving as the "blank" 9. add 1.5mL of Bradford reagent to both tubes, invert several times, and allow for room temp. incubation for 10mins 10. After 10mins, mix and transfer both the "blank" and the "protein BA" into separate cuvettes 11. Take the cuvettes to a spectrophotometer, wipe down the cuvettes using a kim wipe, and zero the spectrophotometer using the "blank" 12. Measure absorbance level at 595nm (**1st time= 0.331A**), zero again, and take a second measurement (**2nd time= 0.330A**) 13. Average the two values (average= 0.3305A) and calculate the protein concentration using y=mx+b y= 1013.9x y= 1013.9(0.3305)= 335.1µg/mL x 2 14. Give the remaining "protein sample" to the TA for storage at -20C
 * y= 670.2µg/mL**

Observations/Deviations: same disposable pestle issue as the RNA Isolation and Extraction experiment and the "protein sample" turned a deep blue, indication a high protein concentration. - I don't think you can conclude that the protein concentration was high just by looking at the color user:emmats

Results for the RNA Isolation and Extraction experiment are still pending Results for the Protein Extraction and Analysis= a protein concentration of **670.2µg/mL**
 * Results:**

At first, a protein concentration of 670.2µg/mL seemed a bit high due to issues of tissue homogenization I had from the disposable pestle but I realized that our tissue sample was from a digestive gland where a high concentration of digestive enzymes are present and enzymes are composed of proteins. Based off of this result, I would like to do another experiment where a tissue sample is taken from a less enzymatic area of the specimen that is also similar in weight and compare the protein concentrations. interesting conclusion! user:emmats
 * Conclusions:**

The purpose of this lab was to expose ourselves to the processes and experiments scientists do, to isolate RNA and protein concentrations from tissue samples for genetic related experiments. The Bradford protein Assay and the line equation y=mx+b was used to measure protein concentrations. It is unclear to me how the Bradford protein assay binds and works to the protein and turning the sample into a blue color. I wish there was more information on what the average protein concentrations are for gills, digestive glands, ect of //Crassostrea gigas// for comparison.
 * Reflections:**


 * Lab Two: RNA Extraction and Protein Analysis, Part Two**


 * Date:** October 11, 2011


 * Summary of the Lab:** Continuing the process of RNA extraction from Lab One with the addition of chloroform and using the nanodrop spectrophotometer for RNA quantification.


 * Materials and Methods:**
 * homogenized tissue sample from Lab One
 * 1000µL and 20µL pipettes
 * 1000µL and 20µL sterile pipette tips
 * sharpie
 * ice buckets
 * RNase free 1.5mL microcentrifuge tubes
 * safety glasses
 * lab coats
 * TriReagent
 * tube rack
 * phenol/chloroform waste containers
 * hot water bath
 * vortex
 * timers
 * nanodrop spectrophotometer
 * isopropanol
 * 75% EtOH
 * 0.1% DEPC treated water
 * RNase free water

1. Incubate homogenized tissue sample from lab one in room temperature for 5 mins 2. add 200µL of chloroform in a fume hood and vortex for 30 seconds 3. once the solution become milky, incubate at room temperature for 5 mins and place in a refrigerated microfuge- spin at max speed for 15mins 4, gently remove the tube to not disturb the contents and carefully transfer the majority of the top clear portion (this is your RNA) to a new tube 5. Dispose the tube containing the organic and interphase liquids 6. add 500µL of isopropanol to the new tube, invert the tube a couple of times and incubate at room temperature for 10mins- the solution should not appear lumpy 7. spin in a refrigerated microfuge at max speed for 8 minutes and a small white pellet (RNA and salts) should be present 8. remove the supermatant, add 1mL of 75% EtOH to the pellet, and vortex briefly 9. spin in the refrigerated microfuge at 7500g for 5 minutes and remove the supermatant 10. spin again for 15 seconds to remove the remaining EtOH 11. leave tube open and allow the pellet to dry at room temperature for 5 minutes 12. add 100µL of 0.1% DEPC water and mix using a pipette 13. place the tube into a water bath set to 55C for 5 minutes 14. remove the tube and flick a few times to mix and place it on ice= stock RNA sample

__RNA Quantification__ 1. set the nanodrop to "nucleic acids" and pipette 2µL of 0.1% DEPC water into the nanodrop pedestal and lower the arm 2. click "blank" to zero the instrument 3. pipette 2µL of your RNA sample onto the nanodrop pedestal and lower the arm and click "measure" 4. record your RNA concentration (ng/µL), A260/280 ratio, and A260/230 ratio 5. raise the arm and wipe off your sample with a kim wipe 6. Stock RNA was labeled as "RNA Gill SC 10/11" 7. Samples are stored at -80C

__Observations/Deviations__ at step 10, the pipette tip was contaminated from the tip waste jar. The contaminated tip was then used to remove the remaining EtOH, which might have affected our RNA quantification results

A260/280= 1.80 A260/230= 2.36 ng/µL= 221.9
 * Results:**

Our A260/280 ratio was within the acceptable RNA quantification range despite possible contamination issues but our ng/µL seemed to be on the lower side compared to other groups.
 * Conclusions:**

The purpose of this lab was to familiarize ourselves in how RNA quantification is achieved using the nanodrop spectrophotometer. The nanodrop uses the Beer- Lambert Law to calculate the RNA concentration and was pleased to see that our A260/280 ratio fell into the acceptable range.
 * Reflections:**


 * Lab Three: Reverse Transcription and primer design**


 * Date: 10/18/2011**


 * Summary of Lab:** performing reverse transcription on our stock RNA to cDNA because cDNA is more stable and can be used for measuring gene expression.


 * Materials and Methods:**
 * stock RNA extracted from lab two
 * 1000µL, 5µL, and 2µL pipettes
 * 1000µL, 5µL, and 2µL pipette tips
 * tip waste jar
 * 0.5mL PCR tubes
 * tube racks
 * kimwipes
 * Lab coat
 * safety glasses
 * gloves
 * ice buckets
 * thermo cycler
 * M-MLV reverse transcriptase
 * M-MLV 5X reaction buffer
 * oglio dT
 * dNTPs
 * DI water

1. stock RNA was placed in -80C so let it incubate at room temperature until it is in a liquid phase and mix the RNA by inverting the tube several times 2. a 0.5mL PCR tube is labeled as "cDNA" 3. add 5µL of the stock DNA 4. 1µL of oglio dT 5. 4µL of DI water 6. incubate the PCR tube for 5 minutes at 70C in the thermocycler 7. after 5 minutes, briefly centrifuge and place on ice for a few minutes 8. add 5µL M-MLV 5X reaction buffer 9. 4µL of DI water 10. incubate the mixture for 60 minutes at 42C 11. heat inactivate for 3 minutes at 70C on the thermocycler 12. spin down the tube and store in -20C

Observations/Deviations: some amount of solutions from steps 3 to 9 were stuck in the pipette tip of the 2µL pipette.


 * Results:** PCR tube was stored in the -20C refrigerator and will continue with the experiment in Lab four


 * Conclusions:** will be continued in Lab four


 * Reflections:** The purpose of this lab was to practice how to perform reverse transcription to synthesize cDNA since it is more stable and can be used to measure gene expression.


 * Lab Four: Tissue dissection, primer reconstitution, end-point PCR**


 * Date: 10/30/2011**


 * Summary of Lab:** Preparing cDNA samples for PCR with the primers we ordered and dissecting/collection tissue samples from our experimental specimens.


 * Materials and Methods:**
 * 1- 1000µL pipettes
 * 1- 1000µL pipette tips
 * tip waste jar
 * tube racks
 * 1.5mL microcentrifuge tubes
 * 0.5mL PCR tubes
 * pH 8.5 TE Buffer
 * cDNA
 * 2x Go Taq
 * dNTPs
 * Primers
 * DI water
 * thermal cycler
 * kimwipes
 * ice buckets
 * lab coats
 * safety glasses
 * gloves


 * Tissue sampling**
 * plastic dissecting trays
 * shucking tools
 * gloves
 * lab coats
 * safety glasses
 * dry ice
 * 1.5mL microcentrifuge tubes
 * 10% bleach
 * 90% ethanol
 * tube racks
 * kimwipes

1. spin down primer for a couple mins and add appropriate amount of pH 8.5 TE buffer ex) 20.4nm= 204µL of TE buffer 2. incubate at 45C for one minute and vortex
 * Making 100µM Stock solution**

1. add 10µL of the 100µM stock solution and 90µL of DI water
 * Making 10µM Stock solution**

- made four PCR samples: two samples with our cDNA and two negative controls labeled NC1 and NC2 1. make PCR master mix by adding: 125µL of 2x GoTaq, 5µL of forward primer **(80477617 HSP70F)**, 5µL of reverse primer **(80477618 HSP70R)**, and 105µL of DI water - we multiplied each amount by five to account for pipetting errors 2. pipette 48µL of the master mix into all four 0.5µL PCR tubes 3. label two tubes as cDNA1 and cDNA2- add 2µL of cDNA to each tube so that the total amount in each tube is 50µL- mix by pipetting 4. label the other two tubes as NC1 and NC2- add 2µL of DI water to each tube so that the total amount in each tube is 50µL- mix by pipetting 5. spin the tubes and place into the thermocycler for a thermo cycling profile
 * Making cDNA-PCR samples: 2x GoTaq master mix already has dNTPs and GoTaq polymerase mixed in**

Denaturation 95C 5min- 1 cycle Denaturation 95C 30sec- 40 cycles Annealing 55C 30sec Extension 72C 90sec Final Extension 72C 3min- 1 cycle Placed in -20C

1. label correct amount of 1.5µL tubes for mantle and gill tissue sampling 2. shuck oysters on the plastic dissecting trays 3. sterilize scissors and tweezers by placing in 10% bleach first then in 90% EtOH 4. take needed tissue sample and place in correct tube and store on dry ice
 * Tissue Sampling**

observations/deviations: control and experimental oysters were mixed up during tissue collecting


 * Results:** four PCR tubes were made for lab five


 * Conclusions:** will be continued in lab five


 * Reflections:** the purpose of this lab was to teach us our to prepare PCR samples for gene expression studies including specific protein expressions and epigenetic studies.


 * Lab Five: Agarose gel, electrophoresis, protein SDS/PAGE, and Western blot**


 * Date: November 1, 2011**


 * Summary of Lab: Agarose gel electrophoresis of amplified PCR products from last week, extracting proteins from tissue samples and running it on a SDS/PAGE gel and on a Western Blot**


 * Materials and Methods**

__**Agarose Gel Electrophoresis**__
 * 1-1000µL pipettes
 * 1-1000µL pipette tips
 * tip waste jar
 * 1.5% agarose gel (already made by TA with 1X TBE buffer, agarose gel powder and ethidium bromide)
 * electrophoresis gel rig
 * kimwipes
 * gloves
 * safety glasses
 * lab coats
 * amplified PCR products from last week's lab
 * 100bp PCR ladder
 * tube racks

1. place the already made agarose gel box already filled with 1X TBE buffer covering the wells 2. load 5µL of 100bp PCR ladder in far left lanes 3. load 25µL of your amplified PCR products into 4 wells (2 wells with cDNA samples and 2 wells as negative controls) 4. place the lid on and run the gel at 100V for an hour 5. after an hour, place gel onto a UV transilluminator


 * __Protein Extraction for SDS/PAGE gel and Western blot__**
 * 1-1000µL pipette
 * 1-1000µL pipette tips
 * tip waste jar
 * kimwipes
 * lab coats
 * safety glasses
 * gloves
 * sharpies
 * 1.5mL snap cap
 * CellLytic MT solution
 * bucket filled with ice
 * refrigerated microfuge
 * tissue sample that was stored in -80C

1. Mantle tissue sample of a dry, diploid //C. gigas-// 0.027g 2. place tissue sample in a 1.5mL tube 3. add 500µL of CellLytic MT solution 4. homogenize tissue with a sterile, disposable pestle 5. close tube and invert it several times 6. spin in a refrigerated microfuge at max speed for 10mins 7. label another tube as "protein" and transfer only the clearish liquid and store on ice


 * __SDS/PAGE- PROTEIN__**
 * 1-1000µL pipettes
 * 1-1000µL pipette tips
 * tip waste jar
 * kimwipes
 * safety glasses
 * lab coats
 * gloves
 * sharpies
 * 1.5mL screw cap tubes
 * tube racks
 * timers
 * water bath
 * tube "floatie"
 * glass beaker with boiling water on a heating block
 * protein gel box-provided
 * power supply
 * tray for gels
 * plastic wrap
 * shaker
 * 2X SDS reducing buffer
 * protein ladder
 * light box

1. in a 1.5mL screw cap, add 15µL of your protein stock and 15µL of 2X reducing sample buffer- place the remaining protein stock in -20C 2. mix by flicking and centrifuge for about 10 seconds 3. boil sample for about 5 minutes 4. after boiling centrifuge got 1 minute 5. load 30µL of your sample into a well 6. put the lid on and run the gel at 150V for 15mins 7. remove gel from the box and crack open the cassette to reach the gel 8. notch a corner for correct orientation

__observations:__ wrong buffer was used the first time-our blue colored sample became clear as soon as it was loaded into the wells supposed to run the gel for 45mins but was reduced to 15mins


 * __Western Blot__**
 * nanopure water
 * gloves
 * safety glasses
 * kimwipe
 * lab coats
 * falcon tube to use as a roller
 * gel staining tray
 * blocking solution
 * primary antibody
 * secondary antibody
 * rotary shaker
 * antibody wash
 * timers
 * chromogenic substrate
 * SDS/PAGE gel
 * tris-glycerine transfer buffer
 * filter paper
 * nitrocellulose membrane

1. soak filter paper, membrane, and gel in tris-glycerine tranfer buffer for 15mins 2. on a semi-dry blotting apparatus assemble the blotting sandwich: anode(+), filter paper, membrane, gel, filter paper cathode(-) 3. use a falcon tube and roll on the sandwich to squeeze any excess liquid 4. transfer and run blot for 30minutes at 20V 5. remove gel from the sandwich and rinse adhered pieces of gel with transfer buffer 6. rinse member twice for 5min with nanopure water 7. place membrane in a plastic box, add 10mL of blocking buffer, cover, and incubate overnight on a rotary shaker at 1rev/sec //Done by TA// 8. decant liquid 9. rinse membrane with 20mL of nanopure water for 5mins, decant, repeat 10. incubate membrane in 10mL primary antibody, decant 11. rinse membrane with 20mL of antibody wash for 5mins, decant, repeat 3 times 12. incubate membrane in 10mL of secondary antibody solution for 30mins, decant 13. rinse membrane with 20mL antibody wash for 5mins, decant, repeat 3 times 14. rinse membrane with 20mL nanopure water for 2mins, decant, repeat 2 times 15. incubate membrane in 5mL of chromogenic substrate until purple bands appear-takes about 1-60mins 16. dry membrane on a clean piece of filter paper in open air

__observations:__ don't know how long the membrane was rinsed with the primary antibody solution

Agarose gel: samples seemed to be around 200bp, negative controls had primer dimers SDS/PAGE-WB: patches of dark and faded bands
 * Results:**


 * Conclusions**: for the agarose gel, having dark, fluorescent bands for our two cDNA samples showed that the protein we were trying to amplify was indeed expressed in the tissue sample. For the western blot, there seems to be contamination because clear, definitive bands did not appear- seemed blotchy.


 * Reflections**: the purpose of this lab was to help experience and practice the different stains to qualitatively confirm the expression of a gene/protein, which will be necessary and needed for our group projects.


 * Lab 6: DNA Extraction with DNazol**


 * Date: November 8, 2011**


 * Summary:** extracting DNA from tissue samples using DNazol for quantification


 * Materials and Methods**
 * 1-1000µL pipettes
 * 1-1000µL pipette tips
 * tip waste jar
 * kim wipes
 * safety glasses
 * lab coats
 * 1.5mL tubes
 * sharpie
 * microcentrifuge at room temperature
 * razor blades
 * tweezors
 * weight scale
 * plastic weigh boats
 * tissue samples
 * plastic pestles
 * 10% bleach
 * 100% ethanol
 * 75% ethanol
 * 95% ethanol
 * 0.1% DEPC water
 * DI water
 * tube racks
 * DNazol
 * Proteinase K
 * sterile 50mL falcon tube
 * Nanodrop

- 31 gill tissue samples: 6 diploid low pH 5 triploid low pH 3 triploid wet 6 diploid wet 5 triploid dry 6 diploid dry

1. each gill tissue was dissected to weigh between 25-50mg, diced up with sterile razor blades and placed in individual 1.5mL labeled tubes 2. a master mix was made in the 50mL falcon tube 0.5mL of DNazol x 34 (used 34 instead of 31 tissue samples for pipette errors)= 17mL of DNazol 2.4µL of ProK x 34= 21.6µL of Prok 3. 15.3µL of the Dnazol/ProK master mix solution was pipetted into each tube 4. sterile plastic pestles were used to homgenize the tissue samples. Used pestles were placed in 10% bleach, then 95% ethanol, and then in DI water for re-use 5. samples were allowed to incubate for an hour at room temperature 6. after an hour, the samples were spun in the centrifuge at 10,000g for 10mins at room temperature 7. new, labeled tubes were made and only the supernatant was transferred 8. 0.5mL of 100% ethanol was added to each tube and each tube was inverted 5-8 times- there should be a white, cloudy precipitate 9. incubate samples at room temperature for 1min 10. spin down the sample in the centrifuge at 5,000g for 5 mins at room temperature 11. let the samples in room temperature for 1min and remove the lysate (liquid) the pellet at the bottom of the tube is your DNA 12. wash your DNA with 1mL of 75% ethanol, invert tubes 6 times, let the samples sit at room temperature, remove ethanol, and repeat 13. remaining ethanol at the end of second wash should be removed with a small pipette 14. add 300µL of 0.1% DEPC water and mix using your pipette to dissolve the DNA 15. DNA samples brought to Nanodrop for quantification (this step was not done due to time) so samples were placed in the -20C refrigerator

2. after step 10, some tubes had a pellet that was a mixture between white precipitate and un-homogenized tissue 3. after step 10, some tubes did not have any white precipitate 4. during step 14, some DNA pellets were not dissolving
 * Observations:** 1. DNazol had a viscous/foamy characteristic resulting in some sample having DNazol bubbles/foam


 * Results:** majority of the samples had white precipitate (DNA) but DNA quantification has yet to be done


 * Conclusions**: DNA quantification using the Nanodrop has not been done yet


 * Reflections:** the purpose of this lab was to help familiarize us with the techniques to extract DNA and see the differences between the techniques we used to extract RNA. DNA extraction is used for the study of epigenetics and DNA methylation for quantitative data. For future purposes, I would do a better homogenization process with the tissue samples by dicing the tissue more thoroughly so that the DNazol digests the tissue more effectively.


 * Lab Seven: Quantitative PCR, DNA extraction, epigenetics**


 * Date: November 15, 2011**


 * Summary:** In this lab, we diluted DNA extracted from oyster, fruit fly, and human samples for dot blotting and chromogenic immunodetection of methylated cytosines in the DNA.


 * Materials and Methods**


 * Methylated cytosine Dot blot**
 * fly DNA
 * 1.5mL screw cap tubes
 * lab coats
 * safety glasses
 * gloves
 * tube racks
 * ice buckets
 * sharpie
 * kimwipe
 * vacuum manifold
 * nylon mmbrane
 * 6X SSC
 * filter paper
 * hot plate
 * large beaker
 * water
 * floaties
 * ice
 * microcentrifuge
 * 1-1000µL pipette
 * 1-1000µL tips
 * scissors
 * plastic wrap
 * denaturation buffer
 * 20X SSC
 * UV transilluminator
 * nanopure water
 * Blocker/Diluent Part A
 * Blocker/Diluent Part B
 * plastic dish with cover
 * rotary shaker
 * blocking solution
 * 5-MeC antibody
 * TBS-T
 * secondary antibody solution
 * chromogenic substrate

1. initial dilution of fly DNA= 6µL of DNA+ 34µL of DI water= 40µL total volume 2. 5 labeled 1.5mL screw cap tubes with target concentration, initials, and "DNA" 3. each tube should have a total volume of 200µL

tube 1= 0.8ng/µL+ 124µL of DI water+ 60µL 20X SSC+ 16µL of diluted DNA sample tube 2= 0.4ng/µL 132 60 8 tube 3= 0.2ng/µL 136 60 4 tube 4= 0.1ng/µL 138 60 2 tube 5= 0.05ng/µL 139 60 1

4. cut nylon membrane to fit 72 wells 5. soak membrane in 6X SSC for 10 mins in tip box 6. cut filter paper to same size as nylon and wet in 6X SSC 7. make manifold with membrane lying on top of filter paper 8. denature DNA in boiling water for 10mins and transfer to ice 9. switch on vacuum and pipette 500µL of 6X SSC in each well 10. spin down DNA 11. pipette all of DNA volume into each well 12. note where each DNA was pipetted into 13. soak filter paper and cut to size in denaturation buffer 14. soak in denaturation buffer for 10mins then neutralization buffer for 5 mins 15. place membranes on dry filter paper and let it dry 16. wrap dryed blot in plastic wrap and place DNA side down on UV transilluminator for 2mins at 120V to immobolize DNA

1. 20mL blocking solution= 14ml ultra filtered water+ 4mL blocker/diluent part a+ 2mL blocker/diluent part b 2. place membrane in 10mL of blocking solution in a covered plastic dish 3. incubate for 30mins on rotary shaker at 1rev/sec 4. decant blocking solution 5. rinse membrane in 20mL of water for 5 mins, decant, repeat 6. 10mL of Primary AB solution 1:5000 dilution= 10mL blocking solution+ 2µL primary AB 7. incubate membrane in 10mL of primary AB solution for 1 hour 8. decant AB and wash membrane in 20mL of TBS-T for 5mins, decant and repeat 3X 9. incubate membrane in 10mL of secondary AB for 30mins, decant 10. wash membrane for 5mins with 20mL of TBS-T, decant and repeat 3X 11. rinse membrane in 20mL of water for 2mins, decant, repeat 2X 12. incubate membrane in 5mL of chromogenic substrate until color begins to develop 13. rinse membrane with 20mL of water for 2mins, decant, and repeat 2X 14. dry membrane on clean filter paper
 * Chromogenic Immunodetection**


 * Quantitative PCR**
 * PCR tubes- white and connected
 * PCR tube caps
 * 1.5mL RNase free tubes
 * nuclease free water
 * filter tips
 * thermal cycler
 * kimwipes
 * 2X immuno master mix
 * SYTO-13 dye
 * tube racks
 * ice buckets
 * timers
 * cDNA samples

1. master mix= 25µL total volume= multiply each amount by 5 to account for pipette errors

2X immomix= 12.5µL final concentration= 1X syto-13 dye= 1µL = 2µM upstream primer 10µm= 1.25µL = 2.5µM downstream primer 10µM= 1.25µL = 2.5µM ultra pure water= 7µL

2. add master mix to each PCR well 3. thaw cDNA samples 4. add 2µL of cDNA in 2 wells 5. add 2µL of DI water in other 2 wells= negative controls 6. cap wells and spin down 7. place PCR strip on ice for thermal cycler

thermal cycler conditions 95C for 10mins 95C for 15secs 72C for 30secs return to step 2 for 39 more times 95C for 10secs


 * observations:** cDNA sample was from another classmate- 2N high pH


 * Results:** for the dot blot and chromogenic immunodetection, if you ook at row 5, you cannot see anything and it is clear compared to the other rows that show faint colored circles. This is because the 5 row was loaded with fly DNA which is known to have no methylated cytosine so there would be no binding of the primary and secondary AB for qualitative observations and results. QPCR results are still in progress


 * Conclusions**: dot blot has validated that there are no methylated cytosines in fly DNA


 * Reflections:** Next time I would like to use DNA from an organisms with methylated cytosines for qualitative results and observations.


 * Lab Eight: Restriction enzyme digest and PCR**


 * Date: 11/21-11/23**


 * Summary:** In this lab I diluted my 2N/3N pH DNA samples to prepare for the addition of HpaII and MspI restriction enzymes. The following day, PCR samples were made and was run on a gel.


 * Materials and Methods:**

//**11/21- diluting DNA samples and adding restriction enzymes**//
 * 1-1000µL pipettes
 * 1-1000µL tips
 * tip waste jar
 * kimwipes
 * lab coat
 * gloves
 * safety glasses
 * tube rack
 * DNA samples
 * nanopure water
 * HpaII enzyme with #1 buffer
 * MspI enzyme with #4 buffer
 * 1.5mL tubes
 * incubator

1. 3N/2N pH DNA samples were diluted to equal 1µg of DNA ex) 3N pH sample 1= 81.4ng/µL which equals to 0.0814µg/µL so 1µg/0.0814= 12.29µL of DNA + x amount of nanopure water to bring total volume to 44µL for HpaII, 44.5µL for MspI, and 50µL for control 2. to all HpaII samples, add 5µL of #4 buffer + 1µL of HpaII enzyme 3. to all MspI samples, add 5µL of #1 buffer + 0.5µL of MspI enzyme 4. to all control samples. no buffer and enzymes are added 5. incubate all samples overnight at 37C

//**11/22- making PCR samples and gel**// 1. HpaII samples were heat stopped for 20min at 65C and MspI samples were heat stopped for 20mins at 80C 2. three separate master mixes were made in 1.5mL tubes because total volume of one master mix would not fit in one 1.5mL tube 3. each group (HpaII, MspI, Control) had 11 tubes so i multiplied each amount needed by 12 to account for pipette errors
 * 1-1000µL pipettes
 * 1-1000µL tips
 * tip waste jar
 * tube rack
 * incubated diluted DNA samples
 * lab coat
 * safety glasses
 * PCR tubes
 * gloves
 * 2X Go Taq
 * C. gigas HSP70 F & R 10µM primers
 * nuclease free water
 * agarose powder
 * PCR chamber
 * 1X TAE buffer
 * ethidium bromide
 * thermal cycler
 * 1.5mL tubes

5X Go Taq: 25µL x 12= 300µL in each 1.5mL tube 10µM Forward primer 1µL x 12= 12µL in each 1.5mL tube 10µM Reverse primer 1µL x 12= 12µL in each 1.5mL tube nuclease free water 21µL x 12= 252µL in each 1.5mL tube DNA 2µL in each PCR tube bringing the total volume to 50µL

4. PCR tubes were placed in thermal cycler denaturation 95C- 5min for 1 cycle denaturation 95C- 30sec for 40 cycles annealing 55C- 30sec extension 72C- 30sec final extension 72C- 3min for 1 cycle hold 4C- infinite

5. 1.5% agarose gel was made with 2g of agarose powder+ 150mL of 1X TAE + 10µL of EtBr 6. two rows of wells were made on gel using two combs that had 20 wells each= 40 wells total 7. gel was wrapped in saran wrap and placed in 20C fridge


 * //11/23- run PCR gel//**
 * 1-1000µL pipettes
 * 1-1000µL tips
 * tips waste jar
 * kimwipe
 * electrophoresis gell chamber filled with TAB buffer
 * 1X TAE
 * lab coat
 * safety glasses
 * UV transilluminator
 * gloves
 * PCR samples
 * 100bp ladder
 * agarose gel from 20C fridge

1. place gel in chamber-make sure all wells are filled with TAB buffer 2. 5µL of ladder was placed in first well of both rows 3. 10µL of sample was placed in appropriate wells 4. gel ran for 45mins at 100V


 * observations:** possible contamination from agarose gel chamber


 * results**: HpaII samples had solid bands not too far from the wells while MspI samples had solid bands and faint bands below the solid bands.


 * conclusions**: There might have been a possible contamination with the MspI samples because faint bands should not appear. Either the primers did not work with the enzymes or the oysters might have had a mutation CCGG to TCGG making it so that it is unrecognizable by the enzymes.


 * Reflections:** Next step starting this week, qPCR will be performed on the same samples to see if we can get different results.


 * Lab Nine: Discussion practice**


 * December 4, 2011**


 * Summary:** we are answering questions to helps us write the discussion sections for our paper

//**1. Detail at least 2 reasons why your results turned out the way they did. This should be easy to do if your results are "unexpected", but even expected results can have multiple explanations. Really think about this, the answer "because I messed up in lab" (or any variation thereof) is not acceptable.**// My results for regular PCR had all bands appearing for both hpaII and mspI enzymes with faint bands appearing under only the mspI samples. This could indicate a possible contamination with the mspI samples or a single nucleotide mutation where a CCGG sequence is a TCGG sequence and the mspI enzyme would not recognize the "T" nucloetide.

For my qPCR results, an incorrect primer was used where the product size of the sequenced primer was around 386bp and my product size was >500bp. user:emmatsI don't think it was an incorrect primer, but I'm still not sure what happened :) You did get an unexpectedly big product size.

//**2. What are two obstacles that you encountered during your lab work and experimental design? Did these obstacles affect your results? Why?**// The main obstacle I encountered was extracting the DNA from the tissue samples because some sample still had undigested tissue at the end of the extraction while other samples did not have a white precipitate at all, making the DNA concentration for my samples scewed.

Another obstacle I encountered was pipetting the correct amount of digestive enzymes because the amount was very small (1µL and 0.5µL) and it seemed as though the pipettes were not calibrated recently. Small amounts of whatever I was pipetting would still remain in the tip.

//**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?**// Although my results were not what was expected, the protocols i followed to make the products for both the regular PCR and qPCR experiments still show that there is something going on in the DNA methylation of the stressed oysters. user:emmatsPlease elaborate on this in your discussion.

//**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?**// My results for qPCR completely stumped me because the graphs were skewed and all over the screen. Next time, I will use the right product size primer of hsp70.

//**5. In about 3 sentences each, summarize 2 papers that you are going to cite in your own paper that give insight into the results that you found.**// One paper is the one written by Mackenzie with professor Roberts on DNA methylation in the pacific oysters, which helped with primer choices and a baseline to compare my results to. Another paper is by Hoffman et. al, about the effects of ocean acidification on calcifying marine organisms, which helped give me a general idea on quantifying and qualitatively seeing gene expressions of a gene in interest. user:emmatsMore information!