Diana

December 3, 2013

Ran qPCR for the third time. Only treatment samples from week 2 were amplified.


 * Copper/ high CO2 || B1 || 55 ||
 * || B2 || 4.11 ||
 * Copper/ low CO2 || C1 || 32 ||
 * || C2 || 1.05 ||

--- November 26, 2013

made cDNA from all 7 samples. Tested primers (qPCR) with negative control (water), B1 and C1.

qPCR 1: Testing of primers was successful. negative control: 82419 B1: 55.13 C1: 32.1


 * Reverse Transcription**
 * 1) Mix stock RNA sample by inverting tube several times
 * 2) 7- 0.5mL PCR tubes (green), labeled: BL, CO1, CO2, B1, C1, B2, C2
 * 3) Add 5µL of RNA + 12.75 µL of master mix (MM1) into each tube
 * 4) MM1 -
 * 5) 4µL - oligo dT (0.5µL x 8)
 * 6) 98µL - nuclease free H2O (12.25µL x 8)
 * 7) Incubate mixture for 5 minutes at 70C in thermocycler, then immediately transfer to ice
 * 8) Add 7.25µL into each tube
 * 9) 40µL - M-MLV 5x Reaction Buffer (5µL x 8)
 * 10) 10µL - dNTP (1.25µL x 8)
 * 11) 4 µL - M-MLV RT (0.5µL x 8)
 * 12) 4µL - nuclease free H2O (4µL x 8)
 * 13) Incubate mixture (25µL total) for 60 minutes at 43C and then heat inactivate at 70C for 3 minutes in the thermacycler
 * 14) Store on ice


 * qPCR**

Materials:
 * 1) PCR plates (white); optically clear caps x 4
 * 2) 1.5 mL microcentrifuge tubes
 * 3) Nuclease free water
 * 4) Filter tips
 * 5) Opticon thermal cycler
 * 6) Kim Wipes
 * 7) 2x Immomix Master Mix
 * 8) SYTO-13 Dye
 * 9) microcentrifuge tube racks
 * 10) ice buckets
 * 11) cDNA samples (32g)
 * 12) HSP70 - primer

Methods: --
 * 1) Prepare a "master mix" (Negative control, B1, C1 + 1 for mistakes):
 * 2) 50 µL - Master Mix (SsoFast EvaGreen supermix) ( 12.5 µL x 4)
 * 3) 2 µL - Upstream primer (10 µM) [HSP70] (0.5 µL x 4)
 * 4) 2 µL - Downstream primer (10 µM) [HSP70] (0.5 µL x 4)
 * 5) 42 µL - Ultrapure water (10.5 µL x 4)
 * 6) Add 25 µL of the "master mix" to each well of the white PCR plate
 * 7) Thaw cDNA samples
 * 8) Once cDNA samples have been thawed, add 1 µL of the sample to **two** of the wells - template
 * 9) Add 1 µL of ultra pure water to **one** of the wells - negative controls
 * 10) Cap the wells securely
 * 11) Label the control and 2 cDNA (B1, C1) templates
 * 12) Place strips on ice
 * 13) Take sample upstairs to qPCR machine, and spin the strips to collect volume at the bottom of the wells
 * 14) Load the PCR plate with the samples, verify the CONDITIONS (below) are correct and start the run
 * 15) CONDITIONS
 * 16) 95C for 10 minutes (denature)
 * 17) 95C for 15 seconds (denature)
 * 18) 55C for 15 seconds (anneal)
 * 19) 72C for 15 seconds + plate read (extend)
 * 20) Repeat step 2 - 39x
 * 21) 95 for 10 seconds
 * 22) Melt curve from 65C - 95C, at 0.5C for 5 seconds (+ plate read)

November 20, 2013

Performed final tissue extractions. 10 mussels (5 from each condition) from high CO2+Cu/ low CO2+Cu died. Total of 5 samples - 1 control, 2 from week one, 2 from week 2 (from Cu treatments only). RNA was isolated from all 5 samples and quantified.


 * || Baseline || Control 1 || Control 2 || B1 || C1 || B2 || C2 ||
 * Absorbance || 6.853 || 15.548 || 12.490 || 10.529 || 19.545 || 14.671 || 14.584 ||
 * 260-10 || 15.740 || 36.519 || 28.174 || 23.686 || 41.591 || 21.679 || 19.917 ||
 * 280-10 || 8.034 || 18.247 || 14.150 || 12.145 || 20.834 || 10.897 || 9.780 ||
 * 260/280 || 1.96 || 2.00 || 1.99 || 1.95 || 2.0 || 1.99 || 2.04 ||
 * 260/230 || 2.30 || 2.35 || 2.26 || 2.25 || 2.13 || 1.48 || 1.37 ||
 * ng/uL || 629.6 || 14.607 || 1127.2 || 974.4 || 1663.7 || 867.2 || 796.7 ||

RNA Isolation:

Materials:
 * 1) Micropipettes (1-1000µL)
 * 2) sterile filter pipette tips (1-1000µL)
 * 3) 1.5 mL microcentrifuge tubes
 * 4) microcentrifuge rack
 * 5) ice bucket
 * 6) cortex
 * 7) hot water bath
 * 8) chloroform
 * 9) RNAase free water
 * 10) chloroform
 * 11) isopropanol
 * 12) 75% ethanol
 * 13) 0.1% DEPC treated water

Methods:

--
 * 1) Heat water bath to 55C
 * 2) Incubate homogenized tissue sample (from freezer) at RT for 5 minutes
 * 3) Under the fume hood add 200µL of chloroform
 * 4) Vortex sample for 30 seconds (the solution should become a milky emulsion)
 * 5) incubate at RT again for 5 minutes
 * 6) Spin sample in centrifuge (refrigerated) at 14,000 x g for 15 minutes
 * 7) CAREFULLY remove sample from centrifuge (do not allow layers/tissue to mix)
 * 8) Transfer the aqueous (clear) phase to a new microcentrifuge tube (DO NOT transfer any of the interphase)
 * 9) Label tube: **RNA, DC, 10/08**
 * 10) Close the tube containing the interphase, and set it aside to be disposed of later
 * 11) Add 500µL of isopropanol to the new "RNA" tube. Mix by inverting the sample multiple times (the solution should not be "viscous/lumpy")
 * 12) Incubate sample at RT for 5 minutes
 * 13) Centrifuge (refrigerator) "RNA" sample for 8 minutes at 14,000 x g. When positioning tube, place the hinge pointing away from the center of the centrifuge
 * 14) Remove sample from centrifuge. A small white pellet should be present
 * 15) Using a pipette, carefully remove supernatant from solution
 * 16) Add 1mL of 75% Ethanol to the pellet.
 * 17) Vortex sample until pellet is dislodged from the side of the tube
 * 18) Spin sample in centrifuge at 7,500g for 5 minutes
 * 19) Remove sample from centrifuge and CAREFULLY remove supernatant
 * 20) Briefly spin sample at 14,000g to pool residual EtOH
 * 21) Using a small pipette tip (P10 or P20), remove remaining EtOH
 * 22) Leave tube open and allow pellet to dry for no more than **5** minutes
 * 23) Add 100µL of 0.1% DEPC-H2O to the sample by pipetting up and down until the pellet is dissolved (vortex if necessary)
 * 24) Incubate sample at 55C (water bath) for 5 minutes to help solubilize RNA
 * 25) Remove tube from heat, and flick a few times to make sure everything is mixed
 * 26) Place sample in freezer at -80C

November 12, 2013

Gill tissues samples were taken from 1 mussel in each treatment conditions. 2 gill samples - one for protein extraction and one for RNA isolation - from each tank. 1 foot tissue sample from the same mussels. Tissue samples are stored at -80C, and are going to be analyzed and processed next week.

November 5 - 6, 2013

4 tanks were set up (2 - pH 7.5 (high)), .5mg/L of Copper were added into 2 tanks (normal+high). 10 mussels were placed in each tank on Wednesday. Week 0 (baseline) tissue samples were collected on Tuesday (11/5) - gill + foot. Gill tissues - 4 tubes (weighing between 0.2-0.5g) kept at -80C. Labeled: date, M1 or M2 (depending on which mussel they came from) and mass of tissue. RNA and protein will be extracted from these samples towards the end of the experiment.

October 29, 2013

Finalized project set up with team. 4 conditions - 1 control (no Cu, 400ppm CO2), no Cu + 1500ppm Co2, Cu+1500ppm CO2, 400ppm + Cu. 10 mussels in each condition, tissue samples extracted every Tuesday during lab (gill and foot), and analyzed using SDS-PAGE and qPCR during weeks 2 and 4 of November. Set up tanks on Friday (11/01) - began bubbling CO2 into them. pH will be measured later on (11/03) to determine if it's within our desired range. Decided to measure overall rate of gene expression of HSP70 in mussels, instead of MT-20. Studies have shown that HSP70 is affected by the presence of metals. And chose to use copper instead because the health risks related to Cadmium are too high. Still unsure about whether or not we are going to be feeding the mussels, but tanks will be changed every Tuesday.

October 22, 2013


 * Protein SDS/PAGE and Western blot, analyze conventional PCR (agarose gel) & qPCR data**


 * Summary:**

Our extracted protein samples were run through SDS-PAGE gels, and then stained to revel the proteins in each lane using a western blot. PCR was done using an agarose gel with our cDNA samples, amplified bands were analyzed under a UV light. qPCR data from last week (10/15) was analyzed during lecture.


 * Materials and Methods:**

SDS-PAGE

Materials:
 * 1) Micropipettes (1-1000µL)
 * 2) sterile filter pipette tips (1-1000µL)
 * 3) sterile gel loading tips
 * 4) 1.5 mL screw cap tubes
 * 5) microcentrifuge tube rack
 * 6) heating block w/ water bath
 * 7) tube "floatie" (water bath)
 * 8) protein gel box
 * 9) SDS-PAGE gels
 * 10) trays for staining gels
 * 11) platform shaker
 * 12) 2x SDS reducing sample buffer
 * 13) protein ladder marker
 * 14) gel running buffer
 * 15) light box

Methods:
 * 1) Boil water on hot plate
 * 2) In a 1.5mL SCREW CAP tube add **15µL** of the extracted protein and **15µL** of 2x reducing sample buffer
 * 3) Mix sample by flocking
 * 4) Label new SCREW CAP: **DC, 10/22**
 * 5) Boil sample in water bath for 5 minutes
 * 6) Gel assembly
 * 7) Remove gel from packaging
 * 8) Remove comb
 * 9) Rinse gel with water
 * 10) Place gels in box, with wells facing towards you, lock in place
 * 11) When sample is finished boiling, load in centrifuge for 1 minute (make sure ladder has been added into well 1)
 * 12) Once samples are all loaded, place lid on box, plug in electrodes
 * 13) Allow gel to run for ~1 hour (or until blue line is on/near the bottom of the gel) **[150V]**
 * 14) ~1 hour later, turn power supply off and disconnect gel box from power supply
 * 15) Remove lid from box
 * 16) Disengage the tension wedge
 * 17) Remove gel from gel box
 * 18) Carefully crack open cassette to expose gel
 * 19) Trim wells using blade
 * 20) In order to remember which gel is which, make a distinct mark on the corner of the gel
 * 21) Run gel through western blot
 * 22) After western blot, stain gel to help visualize bands

Western Blot

Materials:
 * 1) Nanopure water
 * 2) Gel staining tray
 * 3) Blocking solution
 * 4) Rotary shaker
 * 5) Primary antibody solution
 * 6) Antibody wash
 * 7) Secondary antibody solution
 * 8) Chromogenic substrate
 * 9) SDS-PAGE gels
 * 10) Tris-Glycine transfer buffer
 * 11) Filter paper
 * 12) Nitrocellulose membrane
 * 13) Semi-dry transfer station

Methods:
 * 1) Soak the filter paper, membrane and SDS-PAGE gel in Tris-Glyycine Transfer Buffer for 15 minutes
 * 2) Assemble the blotting sandwhich in the semi-dry blotting apparatus:
 * 3) Anode (+++)
 * 4) Filter paper
 * 5) Membrane
 * 6) SDS-PAGE Gel
 * 7) Filter paper
 * 8) Cathode (-)
 * 9) Transfer the blot for 30 minutes at 20V
 * 10) Remove the gel from the sandwhich and rinse off adhering piece of gel with transfer buffer
 * 11) Wash membrane 2 times, for 5 minutes, with 20mL of pure water
 * 12) Place the membrane in the plastic box and add 10mL of Blocking Solution (30 minutes)
 * 13) Decant liquid
 * 14) Rinse the membrane with 20 mL of water for 5 minutes, then decant. Repeat.
 * 15) Incubate the membrane in 10 mL of Primary Antibody Solution. Decant the solution.
 * 16) Rinse the membrane with 20 mL of Antibody Wash for 5 minutes, then decant. Repeat 3 times.
 * 17) Incubate the membrane in 10 mL of Secondary Antibody Solution for 30 minutes. Decant.
 * 18) Wash the membrane for 5 minutes with 20 mL of Antibody wash, then decant. Repeat 3 times.
 * 19) Rinse the membrane with 20 mL of pure water for 2 minutes, then decant. Repeat twice.
 * 20) Incubate the membrane in 5 mL of Chromogenic Substrate until a purple band appears. This will occur between 1-60 minutes after adding the Chromogenic Substrate.
 * 21) Dry the membrane on a clean piece of filter paper to the open air.

PCR

Materials:
 * 1) Agarose gel
 * 2) 1x TAE Buffer
 * 3) UV transilluminator
 * 4) cDNA sample

Methods:
 * 1) Place gel in gel box and fill with 1x TAE Buffer (fully cover wells)
 * 2) Remove combs from wells
 * 3) Load 7 µL 100bp ladder in far left lane (#1)
 * 4) Load 20µL of cDNA sample into gel
 * 5) Freeze left over sample at -20C
 * 6) Run gel at ~100V for ~` hour
 * 7) Visualize the gel on the UV transilluminator


 * Results:**

SDS-PAGE/ Western blot: saw some protein at the top of well 2 in the gel, and trace amounts of protein transferred over onto the membrane during the western blot PCR: saw one lane in the well where my sample was loaded


 * Conclusion:**

SDS-PAGE/Western blot: Since I thought I did my protein extraction correctly, when I ran my sample (84m,Oly) through the gel I was expecting to see some of it transfer onto the membrane, which was exactly what I saw.

PCR: when I ran my cDNA sample through qPCR last week, I only saw one peak on the melting curve (HSP-70 primer) and since I only saw one lane in my agarose gel, I know I did my cDNA extraction correctly.


 * Reflection:**

The purpose of this lab was to show us different ways that we can analzye our tissue samples, to see if the specific protein we're looking for is present and in what quantities. We will then use these skills when we are analyzing our own samples, once we start running our own experiments.

October 15, 2013


 * Quantitative PCR/ Protein Extraction:**


 * Summary:**

qPCR was used to amplify the cDNA sample generated last week (10/8) from sample gill tissue. And protein was extracted from sample muscle tissue.


 * Materials and Methods:**

Quantitative PCR:

Materials:
 * 1) PCR plates (white); optically clear caps x 4
 * 2) 1.5 mL microcentrifuge tubes
 * 3) Nuclease free water
 * 4) Filter tips
 * 5) Opticon thermal cycler
 * 6) Kim Wipes
 * 7) 2x Immomix Master Mix
 * 8) SYTO-13 Dye
 * 9) microcentrifuge tube racks
 * 10) ice buckets
 * 11) cDNA samples (32g)
 * 12) HSP70 - primer

Methods: Protein Extraction and Analysis: Part 1
 * 1) Prepare a "master mix" (make enough for the number of reactions + 1 for mistakes):
 * 2) 62.5 µL - Master Mix (SsoFast EvaGreen supermix) ( 12.5 µL x 5)
 * 3) 2.5 µL - Upstream primer (10 µM) [HSP70] (0.5 µL x 5)
 * 4) 2.5 µL - Downstream primer (10 µM) [HSP70] (0.5 µL x 5)
 * 5) 51.5 µL - Ultrapure water (10.5 µL x 5)
 * 6) Add 24 µL of the "master mix" to each well of the white PCR plate
 * 7) Thaw cDNA samples
 * 8) Once cDNA samples have been thawed, add 1 µL of the sample to **two** of the wells - template
 * 9) Add 1 µL of ultra pure water to **two** of the wells - negative controls
 * 10) Cap the wells securely
 * 11) Label the 2 controls and 2 cDNA templates
 * 12) Place strips on ice
 * 13) Take sample upstairs to qPCR machine, and spin the strips to collect volume at the bottom of the wells
 * 14) Load the PCR plate with the samples, verify the CONDITIONS (below) are correct and start the run
 * 15) CONDITIONS
 * 16) 95C for 10 minutes (denature)
 * 17) 95C for 15 seconds (denature)
 * 18) 55C for 15 seconds (anneal)
 * 19) 72C for 15 seconds + plate read (extend)
 * 20) Repeat step 2 - 39x
 * 21) 95 for 10 seconds
 * 22) Melt curve from 65C - 95C, at 0.5C for 5 seconds (+ plate read)

Materials:
 * 1) Micropipettes (1-1000µL)
 * 2) sterile filter pipette tips (1-1000µL)
 * 3) 1.5 mL microcentrifuge tubes
 * 4) sterile 2mL screw cap centrifuge tube
 * 5) sterile disposable pipettes
 * 6) microcentrifuge (refrigerated)
 * 7) CellLytic MT Cell Lysis Reagent (with Protease Inhibitor Cocktail added)

Method:
 * 1) Retrieve frozen tissue sample
 * 2) Label tube with sample: **DC, 10/15, 84m Oly**
 * 3) Add 500µL of CellLytic MT solution to the 1.5mL snap cap tube containing your cut piece of frozen tissue
 * 4) Homogenize tissue using a sterile disposable pipette
 * 5) Once fully homogenized, close the tube and invert several times
 * 6) Centrifuge sample (fridge) for 10 minutes at **max** speed
 * 7) While spinning, label a new tube: **Protein, DC, 10/15, 84m Oly**
 * 8) Remove sample from centrifuge
 * 9) Extract centrifuge and place supernatant in "Protein" tube
 * 10) Store sample on ice for next week


 * Results/Conclusion:**

Since we did not quantify our protein samples, I am assuming that my protein extraction was successful, and would have generated a linear graph.

qPCR results: sample 1 (cDNA) = successfully amplified, sample 1 and 2 (cDNA) = 1 peak on melting curve for both (not sure why...)
 * Ct = 35.47 (cDNA) = 5.63
 * Ct = 36.88 (cDNA) = 2.12
 * Ct = 16.86 (control)
 * Ct = 32.13 (control)
 * Arbitrary expression value =10^(-(0.3012* //Ct //)+11.434)


 * Reflection:**

The purpose of this lab is to show how we can use primers to amplify a specific sequence we are looking at, so that when we start working on our own experiments, we'll be able to use qPCR to analyze the gene we're looking at, and know if our cDNA had the specific sequence/protein we're looking for. And we learned how to extract protein from tissue samples so that we can use the same technique with our tissue samples later on in the quarter.

We finalized our group projects: metallothionein and ocean acidification [Mussels] - (Charlie, Rachel, Matt)

October 8, 2013


 * RNA Extraction Part 2:**


 * Summary:**

RNA was completely isolated from a sample of gill tissue that was prepared with 1,000µL TriReagent. The extracted RNA was then used to generate a cDNA copy.


 * Materials and Methods:**

RNA Isolation:

Materials:
 * 1) Micropipettes (1-1000µL)
 * 2) sterile filter pipette tips (1-1000µL)
 * 3) 1.5 mL microcentrifuge tubes
 * 4) microcentrifuge rack
 * 5) ice bucket
 * 6) cortex
 * 7) hot water bath
 * 8) chloroform
 * 9) RNAase free water
 * 10) chloroform
 * 11) isopropanol
 * 12) 75% ethanol
 * 13) 0.1% DEPC treated water

Methods:


 * 1) Heat water bath to 55C
 * 2) Incubate homogenized tissue sample (from freezer) at RT for 5 minutes
 * 3) Under the fume hood add 200µL of chloroform
 * 4) Vortex sample for 30 seconds (the solution should become a milky emulsion)
 * 5) incubate at RT again for 5 minutes
 * 6) Spin sample in centrifuge (refrigerated) at 14,000 x g for 15 minutes
 * 7) CAREFULLY remove sample from centrifuge (do not allow layers/tissue to mix)
 * 8) Transfer the aqueous (clear) phase to a new microcentrifuge tube (DO NOT transfer any of the interphase)
 * 9) Label tube: **RNA, DC, 10/08**
 * 10) Close the tube containing the interphase, and set it aside to be disposed of later
 * 11) Add 500µL of isopropanol to the new "RNA" tube. Mix by inverting the sample multiple times (the solution should not be "viscous/lumpy")
 * 12) Incubate sample at RT for 5 minutes
 * 13) Centrifuge (refrigerator) "RNA" sample for 8 minutes at 14,000 x g. When positioning tube, place the hinge pointing away from the center of the centrifuge
 * 14) Remove sample from centrifuge. A small white pellet should be present
 * 15) Using a pipette, carefully remove supernatant from solution
 * 16) Add 1mL of 75% Ethanol to the pellet.
 * 17) Vortex sample until pellet is dislodged from the side of the tube
 * 18) Spin sample in centrifuge at 7,500g for 5 minutes
 * 19) Remove sample from centrifuge and CAREFULLY remove supernatant
 * 20) Briefly spin sample at 14,000g to pool residual EtOH
 * 21) Using a small pipette tip (P10 or P20), remove remaining EtOH
 * 22) Leave tube open and allow pellet to dry for no more than **5** minutes
 * 23) Add 100µL of 0.1% DEPC-H2O to the sample by pipetting up and down until the pellet is dissolved (vortex if necessary)
 * 24) Incubate sample at 55C (water bath) for 5 minutes to help solubilize RNA
 * 25) Remove tube from heat, and flick a few times to make sure everything is mixed
 * 26) Place sample in freezer at -80C

Reverse Transcription (cDNA):

Materials:
 * 1) Micropipettes (1-1000µL)
 * 2) Sterile filter pipette tips (1-1000µL)
 * 3) RNA samples
 * 4) PCR tubes (0.5mL)
 * 5) Oligo dT
 * 6) Nuclease Free Water
 * 7) Buffer
 * 8) dNTP's
 * 9) M-MLV-Reverse Transcriptase
 * 10) PCR tube rack

Method:


 * 1) Label a 0.5mL PCR test tube: **DC, RNA, 10/08, 32g**
 * 2) Add 5µL of your RNA sample into the 0.5mL tube
 * 3) Add 1µL of oligo dT to the same tube
 * 4) Add 4µL of nuclease free water the new RNA tube
 * 5) Incubate sample at 70C for 5 minutes
 * 6) Remove sample from incubator, and add 14µL of the "Master Mix" (Buffer, NTP's, M-MLV-RT, Nuclease free water) to the tube
 * 7) Invert tube a few times to mix the sample
 * 8) Store sample on ice


 * Results/Conclusion:**

Since we did not perform the RNA quantification, I don't know how pure my RNA sample is. But assuming that it falls within the 1.8-2.0 (A260/A280), I would say that I ended up with a pretty pure RNA sample, that was later used to generate it's cDNA which is more stable and can be used to measure gene expression.


 * Reflection:**

The purpose of this lab is to show us how we can isolate RNA, and then use that sample to generate a copy of it's cDNA which will become very important when we are performing our own experiment. RNA is very unstable, and we can in turn use it's cDNA copy, which is far more stable, in order to analyze things such as gene expression and mutations. We can also run the cDNA through PCR, and examine a specific sequence of the gene that we are looking for.

October 1, 2013


 * DNA and RNA (initiation) isolation**


 * Summary:**

RNA was isolated from a sample of Pacific oyster gill tissue using TriReagent, consisting of guanidine isothiocyanate, phenol and pH, in order to separate RNA from other cellular components. I then used DNAzol to isolate DNA from a sample of muscle tissue (Pacific), which was then quantified using the Nanodrop.


 * Materials and Methods:**

//RNA Extraction://

Materials: Method:
 * 1) Micropipettes (1-1000µL)
 * 2) sterile filter pipette tips (1-1000µL)
 * 3) Sterile (RNAase free) 1.5mL microcentrifuge tubes
 * 4) sterile disposable pestles
 * 5) vortex
 * 6) ice buckets
 * 7) TriReagent
 * 1) Label snap cap tube with tissue sample: **32g, Pac, DC, 10/01/13**
 * 2) Add 500µL TriReagent to the sample, under the fume hood
 * 3) Carefully homogenize sample using sterile pestle
 * 4) After the sample is completely homogenized, vortex for15 seconds and store at **-80C**

//DNA Isolation (DNAzol)//

Materials:
 * 1) Micropipettes (1-1000µL)
 * 2) sterile filter pipette tips (1-1000µL)
 * 3) Sterile 1.5mL microcentrifuge tubes
 * 4) sterile disposable pestles
 * 5) microcentrifuge tube rack
 * 6) microcentrifuge (room temperature)
 * 7) vortex
 * 8) DNAzol
 * 9) 100% ethanol
 * 10) 75% ethanol
 * 11) 0.1% DEPC water
 * 12) KimWipes
 * 13) Nanodrop

Method:
 * 1) Label snap cap tube containing sample: **82m, Oly, DC, 10/01/13** and store on ice
 * 2) Add 500µL of DNAzol to the sample and carefully homogenize using a sterile pestle
 * 3) Add an addition 500µL of DNAzol was then added to the sample and mix once more
 * 4) Let the sample incubate at room temperature for 5 minutes
 * 5) Then centrifuge the sample at 10,000 x g for 10 minutes
 * 6) Remove sample from centrifuge, and transfer supernatant to new tube labeled: **super-DNA,DC**
 * 7) Add 500µL of 100% ethanol to the 'super-DNA' sample, and invert the tube 5-8 times
 * 8) Spin the sample ('super-DNA) in the centrifuge at 10,000 x g for 2 minutes to form a DNA pellet
 * 9) Remove sample from centrifuge, and allow it to sit at room temperature for 1 minute
 * 10) Pipette out the lysate in the sample
 * 11) Wash the DNA with 1mL of 75% ethanol, invert test tube 5-6 times
 * 12) Allow sample to sit at room temperature for 1 minute
 * 13) Remove ethanol from the tube ('super-DNA')
 * 14) Repeat the ethanol wash once more (steps11-13)
 * 15) Remove trace amounts of ethanol left in the tube at the end of the second wash using a 20µL pipette
 * 16) Add 300 µL of 0.1% DEPC water to the DNA sample and pipette up and down multiple times to dissolve the pellet
 * 17) Bring the sample upstairs to the nanodrop

//DNA Quantification://

Method:
 * 1) Pipette 2µL of the DNA sample ('super-DNA') onto the Nanodrop pedestal
 * 2) Lower the arm and click "Measure"
 * 3) Record the DNA concentration (µg/µL), A260/280 ratio and A260/230 ratio
 * 4) Raise the arm and wipe off the sample using a KimWipe
 * 5) Re-label test tube: **DNA,** **82m, Oly, DC, 10/01/13**
 * 6) Store sample at -20C


 * Results:**

A-260-10mm path: 3.236 A-280-10mm path: 1.742 DNA Quantification results: DNA concentration: **161.8µg/µL** A260/280: **1.86** A260/230: **1.08**


 * Conclusion:**

Since purified DNA has an A260/A280 ratio range of 1.7-1.9, my results were as expected because I ended with a A260/A280 ratio of 1.86, which indicates good quality DNA extraction. Based on my results, if I were to continue using this sample I could run it through a gel after adding restriction enzymes to cut it. I could also use it to generate a primer in order to isolate a specific fragment of the DNA using PCR.


 * Reflections:**

The purpose of this lab was to stress the importance of DNA and how everything revolves around it. We can use it to study how the environment influences the genes that are present in and organism, and use it to see how tings change over time. The procedures we used in lab this week were to provide us with skills regarding the isolation of DNA from a tissue sample. And we will then apply some of these skills when we isolate RNA from a different sample next week


 * Notes:**

Oly = Olympia oyster Pac = Pacific oyster


 * Genes:**

Thromboxane synthase: TXA2 - to see how it's regulation could effect hemostasis. F5 and mutations related to it