Joelle's+Notebook

__**Friday 12/ 5 **__  __**Tuesday 12/2**__ Completed captions for supplemental images to accompany the completed individual growth summary. Populations that had no survivors after one year were excluded from the daily growth rate and average net growth. Brent showed me how to run an ANOVA test between each oyster population at different locations and oyster populations within a given site to test the significance of the average net growth difference. He refereed me Vassarstas to learn more about what an ANOVA test really means as well at to look at the PostDoc test to potentially do some further analysis on this data. Overall, It appears that Manchester had the most surviving individuals but Oyster Bay had the greatest daily growth rate and average net growth overall.  __**Monday 12/1**__ Completed measuring Oyster Bay oyster population on ImageJ from when trays were installed in location on 8/18/13 and the same population on 9/19/14. These measurements were converted and the difference between the later measurements and initial measurements was taken divided by the difference in days (426) to calculate the daily growth rate for each individual oyster which was then average to get that trays daily average growth rate so that we can compare growth rate among populations. Still need to finish captions for supplemental images and need to compile individual growth summary.  Completed measuring Fidalgo Bay oyster population on ImageJ from when trays were installed in location on 8/19/14 and the same population on 10/17/14. These measurements were converted and the difference between the later measurements and initial measurements was taken divided by the difference in days (426) to calculate the daily growth rate for each individual oyster which was then average to get that trays daily average growth rate so that we can compare growth rate among populations. Began setting up images and google excel sheet for Oyster Bay population for the same growth rate measurements.  Completed measuring Manchester oyster population on ImageJ from when trays were installed in location on 8/28/14 and the same population on 10/24/14. These measurements were converted and the difference between the later measurements and initial measurements was taken divided by the difference in days (428) to calculate the daily growth rate for each individual oyster which was then average to get that trays daily average growth rate so that we can compare growth rate among populations. Began setting up images and google excel sheet for Fidalgo Bay population for the same growth rate measurements.  Continued measuring Manchester oyster population on ImageJ from when trays were installed in location on 8/28/14 and the same population on 10/24/14. These measurements were converted and the difference between the later measurements and initial measurements was taken divided by the difference in days (428) to calculate the daily growth rate for each individual oyster which was then average to get that trays daily average growth rate so that we can compare growth rate among populations.  Began measuring Manchester oyster population on ImageJ from when trays were installed in location on 8/28/14 and the same population on 10/24/14. These measurements were converted and the difference between the later measurements and initial measurements was taken divided by the difference in days (428) to calculated the daily growth rate for each individual oyster which was then average to get that trays daily average growth rate so that we can compare growth rate among populations.  Completed measuring oysters for size at mortality measurements which is located on google drive. No statistical analysis has been completed on this data sheet yet.  Installed ImageJ on my personal computer and completed ImageJ analysis of Oyster Bay oysters. Jake has gone through Manchester, Fidalgo, and Oyster Bay samples and converted measurements for further statistical analysis.  Completed ImageJ data collection on Fidalgo Bay Oysters.  Continued ImageJ data collection on Fidalgo Bay Oysters.  Completed ImageJ data collection on Manchester bay oysters and started ImageJ data collection on Fidalgo Bay oysters. Both are compiled on spreadsheets on Google Doc shared with Jake that are going to be converted and used for R analysis later on. I continued to measure shell size at mortality from sample 1H 5-8 on 5/23/14 and completed those oysters specifically and still have two gallon sized ziplock bags left to process.  Learned how to use ImageJ from Jake and started taking measurements from trays at Manchester. A Google Doc was created that measurements are being entered into and shared between Jake and I. Today I got through 6 trays so Monday I will need to finish the 6 other trays. Data entered includes sample, area, mean, min, max, angle, and length which were formulated by ImageJ. The first tray I measured was hard to identify the beginning and end of some oysters so I took a picture and circled the oysters that were not measured. Otherwise all other pictures seemed fine but the oysters appear to have stained some of the tiles so finding a definite end to the oyster shell is slightly difficult. Overall, majority of the oysters are measurable.  Empty oyster shells from Jake's project this summer were kept, some frozen. Shell width from the umbo to the loargest diameter of the shell were measured using calipers. Data was enter into size at mortality google doc which included date, site, tray, sample, and size.  Data from Jake's field notebook of year 1 sampling from Oyster Bay, Fidalgo Bay, Manchester, and Dahob were entered into a google doc including the date, site, tray, sample, size and weight for further analysis on R.  __**Monday 10/20**__ -Read through QIAGEN DNeasy Handbook and Commercial Guidelines for Genomic DNA Extraction -Summarized Protocols for both Procedures
 * __Tuesday 11/25__**
 * __Monday 11/24__**
 * __Friday 11/21__**
 * __Tuesday 11/18__**
 * __Monday 11/17__**
 * __ Monday 11/10 __**
 * __Friday 11/7__**
 * __Tuesday 11/4__**
 * __Monday 11/3__**
 * __ Friday 10/31 __**
 * __ Tuesday 10/28 __**
 * __Monday 10/27__**

__//**QIAGEN DNeasy Protocol: Purification of Total DNA from Animal Tissues (Spin-Column Protocol)**//__ -Make sure there is no precipitate in Buffer AL and ATL, if so warm to 56 °C to dissolve -Add appropriate amount of ethanol (96-100%) to Buffer AW1 and AW2 indicated on bottles -Preheat thermomixer to 56 °C -Thaw frozen tissues -Cut tissue into small pieces and place in 15ml microcentrifuge tube (use appropriate starting tissue amounts stated in manufacturer handbook for Qiagen Blood &Tissue Kit) -Add 180 µl Buffer ATL -Add 20 µl proteinase K and vortex -Incubate at 56 °C in thermomixer until completely lysed (1-3 hours depending on tissue) - Vortex and add 200 µl Buffer AL -Vortex again, add 200 µl ethanol (96–100%), and vortex a final time to ensure proper mixing -Pipet all the mixture into the DNeasy Mini Spin Column -Centrifuge at 6000 x g(8000 rpm) for 1 min and discard flow through -Add 500 µl Buffer AW1 and centrifuge for 1 min at 6000 x g (8000 rpm) then discard flow through -Add 500 µl Buffer AW2 and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane -Discard flow through -Place spin column in clean tube and add 200 µl Buffer AE -Incubate at room temperature for 1 min -Centrifuge for 1 min at 6000 x g (8000 rpm) to elute -Elute multiple times to increase DNA yield (optional) otherwise DNA is isolated
 * Preparation**
 * Procedure**

__//**DNAzol® Genomic DNA Isolation Reagant Protocol**//__
 * LYSIS \ HOMOGENIZATION:**
 * Add 1 ml DNAzol + 25 - 50 mg tissue in homogenizer
 * Homogenize tissues in a hand held glass-Teflon homogenizer by applying as few strokes as possible
 * Soft tissues can be homogenized by mixing in pipet
 * CENTRIFUGATION (Optional):**
 * Centrifuge at 10,000 g for 10 minutes
 * DNA PRECIPITATION:**
 * Add 0.5 ml of 100% ethanol per 1 ml of DNAzol used in lysis step
 * Invert tubes 5-8 times for 1-3 minutes to mix
 * DNA should start to become visible as cloudy precipitate
 * Spool visible DNA on pipet tip and transfer to new microcentrifuge tube
 * Let stand for 1 minute and remove remaining DNA precipitate for DNA wash
 * DNA WASH:**
 * Wash twice with 1 ml of 75% ethanol
 * o Wash by inverting the tube3-6 times
 * Allow DNA to settle at bottom
 * Remove remaining ethanol/alcohol
 * DNA SOLUBILIZATION:**
 * Dissolve DNA by adding about 0.2 - 0.3 ml of 8 mM NaOH or water for 20mg of tissue
 * Slowly pass the pellet through a pipette to dissolve
 * Aim for DNA concentration of 0.2 - 0.3 µg/µl

__**Tuesday 10/14**__ -Continued lab orientation with Sam going through official lab checklist -Set up Wiki membership and Google Calendar -DNA Isolation - Olympia Oyster Populations for RAD Sequencing -Observed Sam adding proteinase K to Oly oyster tissues for cell lysis according to Qiagen protocols for DNA preparation and isolation

__**Monday 10/13/14**__ -Lab Orientation with Sam -DNA Isolation - Olympia Oyster Populations for RAD Sequencing -Aided in tissue extraction from 32 randomly selected Olympia Oysters from 3 separate populations (HL, NF, and SN) which were placed in a 96 well DNeasy Blood and Tissue Kit (Qiagen) with Sam and Professor Roberts.