4 23 2015 Heat/Mechanical Shock 24 Hour Time Point

Today I completed the 24 hour time point collection and the collection of the controls for the experiment. Tonight I ran out of liquid nitrogen before doing the control groups. I put them on ice and placed them in the -80 ASAP.

Heat Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml screw cap tube dropped into liquid nitrogen -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Mechanical Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml tube dropped into liquid nitrogen - then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Controls

For controls sample 8 from each group, this should be done on day 2 as less things are going on. Protocol is the same. These oyster should have remained in holding tank-throughout and should simply be removed and sampled. There is no treatment. Using sterile techniqes.

• ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
• 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
• mantle sample (50-100 mg) in 1.5 ml tube dropped into liquid nitrogen then to -80°C
• remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
• shells placed in labeled plastic bags so correspond to sample including oyster id

4 22 2015 Heat/Mechanical Shock Experiment

Today I ran the Heat and Mechanical Shock experiment on my oysters. While most groups got liquid nitrogen treatment, the mechanical H and S groups did not due to lack of Liquid N2. For those samples I placed them on ice and moved them to the -80 as soon as possible.  The procedure went as follows.

Pre-Experiment

Oysters

• 8°C
• fed daily with 5-10 ml per tank commercial shellfish diet (Isochrysis 1800 mix Marine Algae)
• Partial water changes once per week (remove 5 gallons, replace with fresh 5 gallons)
  • Seawater from Seattle Aquarium
• Aeration with aquarium aerator/stone
• circulation with underwater pump
• Tanks 10 gallon plastic storage tubs (costco storage tubs)

Homogenization tubes for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)

Collection 1.5 ml tubes (screw cap) for ctenidia prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)

Collection 1.5 ml tubes (screw cap) for mantle prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, oyster # (1,2,3...), and tissue type (C, M)

15 ml conical tubes for whole oyster prelabelled with date (42215,42315), pop (N,H,S), treatment (T,M) and time point (1, 24) or Control, and oyster # (1,2,3...)

Warm fresh seawater in 500 ml beaker to 38°C by immersing it in a water bath at 38°C.

Experiment

Heat Stress

• 22 oysters each pop in a mesh bag were lowered (stagger each pop by 1 hour) into 500 ml of prewarmed 38°C sea water (1 hr timer started)
  
  • N pop first, H pop next, S pop last
• after 1 hour exposure heat stress ends, animals returned to 8°C water.
• 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
  • 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells in individual labeled ziploc bags so correspond to sample including oyster id

Mechanical Stress

• 22 oysters from 1 pop placed in bucket in centrifuge and spun for 5 minutes at 1000 rpm.  Samples staggered by 1 hour. Completed after all heat shock treatments and samples were taken.
  • N pop first, H pop next, S pop last
• after 5 minutes of mechanical stress, animals returned to 8°C water
• 1 hour post exposure 8 animals sampled from each group staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml Trizol stored at -80°C
  • 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample (decent size) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells in individual labeled ziploc bags so correspond to sample including oyster id

4 2 2015 EZNA Seed Isolation with Fidalgo pt 2.

Today I completed the 96 seed isolation with the EZNA kit. I isolated the last 12 samples for Fidalgo. The samples are in the freezer in 209 and are ready for GBS processing. You can read about the last Fidalgo isolation in this blogpost.

Before using the EZNA Kit I dissected out whole body tissue from 12 seed oysters into the homogenization tube. The oysters are labeled NF 21-32. I used flame sterilized equipment to dissect the animals. 

The protocol is as follows:

1. Added 350 ul ML1 Buffer
2. Added 25 ul Proteinase K solution
3. Used pestle in homogenization tube to grind tissue in solution
4. Vortexed
5. Incubated at 60 C for 30 minutes
6. Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
7. Vortexed
8. Centrifuged 10,000 g for 2 minutes
9. Transferred the upper aqueous phase to new tube (~300 ul)
10. Added 300 ul MBL Buffer
11. Added 10 ul RNase A
12. Vortexed for 15 seconds
13. Incubated at 70C (started at 67.5 C) for 10 minutes
14. Cooled to room temperature sitting for 5 minutes
15. Added 300 ul 100% EtOH
16. Vortexed for 15 seconds
17. Put spin column in collection tube
18. Added 750 ul sample solution to column
19. Centrifuged at 10,000 g for 1 minute at 4C
20. Discarded flowthrough
21. Repeated 18-20 with remaining sample
22. Discarded collection tube and replaced with a new one. 
23. Added 500 ul HBC solution. 
24. Centrifuged at 10,000 g for 30 seconds at 4C
25. Discarded flowthrough
26. Added 700 ul DNA Wash Buffer
27. Centrifuged at 10,000 g for 1 minute at 4C
28. Discarded flowthrough
29. Repeated 26-28
30. Centrifuged Empty column for 2 minutes at 10,000 g at 4C
31. Discarded collection tube and put column into microcentrifuge tube for sample collection
32. Added 100 ul preheated 70C Elution Buffer
33. Incubated for 2 minutes
34. Centrifuged at 10,000 g for 1 minute at 4C
35. Repeated 32-34. 
36. Stored DNA at -20 C

DNA is in a box labelled:

Seed Oly DNA HL NF

3/23/15 Jake Heare 1 of 2

Seed Oly DNA SN 

3/31/15 JH 2 of 2

3 24 2015 EZNA with Fidalgo Seed Oysters

Today I completed isolation of 20 seed oysters from the Fidalgo population using the EZNA extraction kit. This is the same kit I used yesterday. I also ran a gel on those samples and found that even though there was more HMW DNA there was still a lot of degradation in the samples. You can read about it in my other blogpost today. I expect the same results from these samples. I will run a gel on them when I get back next week to check the quality of the extraction. Right now everything is stored in the -20 C Freezer in 209 in a box labeled Seed Oly Extraction 3/23/2015. 

Before using the EZNA Kit I dissected out whole body tissue from 20 seed oysters into the homogenization tube. The oysters are labeled NF 1-20. I used flame sterilized equipment to dissect the animals. 

The protocol is as follows:

1. Added 350 ul ML1 Buffer
2. Added 25 ul Proteinase K solution
3. Used pestle in homogenization tube to grind tissue in solution
4. Vortexed
5. Incubated at 60 C for 30 minutes
6. Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
7. Vortexed
8. Centrifuged 10,000 g for 2 minutes
9. Transferred the upper aqueous phase to new tube (~300 ul)
10. Added 300 ul MBL Buffer
11. Added 10 ul RNase A
12. Vortexed for 15 seconds
13. Incubated at 70C (started at 67.5 C) for 10 minutes
14. Cooled to room temperature sitting for 5 minutes
15. Added 300 ul 100% EtOH
16. Vortexed for 15 seconds
17. Put spin column in collection tube
18. Added 750 ul sample solution to column
19. Centrifuged at 10,000 g for 1 minute at 4C
20. Discarded flowthrough
21. Repeated 18-20 with remaining sample
22. Discarded collection tube and replaced with a new one. 
23. Added 500 ul HBC solution. 
24. Centrifuged at 10,000 g for 30 seconds at 4C
25. Discarded flowthrough
26. Added 700 ul DNA Wash Buffer
27. Centrifuged at 10,000 g for 1 minute at 4C
28. Discarded flowthrough
29. Repeated 26-28
30. Centrifuged Empty column for 2 minutes at 10,000 g at 4C
31. Discarded collection tube and put column into microcentrifuge tube for sample collection
32. Added 100 ul preheated 70C Elution Buffer
33. Incubated for 2 minutes
34. Centrifuged at 10,000 g for 1 minute at 4C
35. Repeated 32-34. 
36. Stored DNA at -20 C

10 28 2014 Updated Fidalgo Temperatures Year 1

FidalgoTempConcatScript.R

Jake H

Tue Oct 28 16:47:12 2014

library(plyr)
library(ggplot2)
library(scales)
fidaugfeb<-read.table('FidAugtoFeb.csv', row.names=1)
#read in data, first change column names and remove data name in excel to make it work
head(fidaugfeb)

##          V2    V3    V4
## 1 8/17/2013  9:51 21.38
## 2 8/17/2013 10:06 21.57
## 3 8/17/2013 10:21 21.57
## 4 8/17/2013 10:36 21.76
## 5 8/17/2013 10:51 22.05
## 6 8/17/2013 11:06 22.05

fidaugfeb<-rename(fidaugfeb, c("V2"="Date",'V3'='Time','V4'='Temp'))
#rename columns
fidaugfeb$Date<-as.Date(fidaugfeb$Date, "%m/%d/%Y")
#tell R that these are dates
tmptst<-ddply(fidaugfeb,.(Date),summarise, mean_temp=mean(Temp,na.rm=T))
#creates mean temperature per date using summary statistics and ddply
plot(mean_temp~Date,data=tmptst)

fidfebmay<-read.table('FidalgoFebtoMay2014.csv', row.names=1)
fidmayjun<-read.table('FidalgoMaytoJun14.csv', row.names=1)
fidjunjul<-read.table('FidalgoJuntoJul14.csv', row.names=1)
fidjuloct<-read.table('FidalgoJultoOct2014.csv', row.names=1)
fidjunoct<-read.table('FidalgoJuntoOct2014.csv', row.names=1)
#entered in all other CSVs
fidfebmay<-rename(fidfebmay, c("V2"="Date",'V3'='Time','V4'='Temp'))
fidmayjun<-rename(fidmayjun, c("V2"="Date",'V3'='Time','V4'='Temp'))
fidjunjul<-rename(fidjunjul, c("V2"="Date",'V3'='Time','V4'='Temp'))
fidjunoct<-rename(fidjunoct, c("V2"="Date",'V3'='Time','V4'='Temp'))
fidjuloct<-rename(fidjuloct, c("V2"="Date",'V3'='Time','V4'='Temp'))
#rename columns
fidfebmay$Date<-as.Date(fidfebmay$Date, "%m/%d/%Y")
fidmayjun$Date<-as.Date(fidmayjun$Date, "%m/%d/%Y")
fidjunjul$Date<-as.Date(fidjunjul$Date, "%m/%d/%Y")
fidjunoct$Date<-as.Date(fidjunoct$Date, "%m/%d/%Y")
fidjuloct$Date<-as.Date(fidjuloct$Date, "%m/%d/%Y")
#Date column as dates
fidy1<-rbind(fidaugfeb,fidfebmay,fidmayjun,fidjunjul,fidjunoct,fidjuloct)
#concats all temp data

fidy1edit<-read.csv("fidY1.csv")
#reads in edited CSV to correct for outliers.
fidy1edit$Date<-as.Date(fidy1edit$Date, "%m/%d/%Y")
#Dates as DATES in r
fidy1editv2<-rbind(fidy1edit,fidjunoct,fidjuloct)
#adds data for June and July to October to preedited temp.
fidy1editv2$Date<-as.Date(fidy1editv2$Date)
#turns dates into dates in r

fidy1v3<-read.csv("fidy1v3.csv")
#reads in editted temp file
fidy1v3$Date<-as.Date(fidy1v3$Date,"%m/%d/%Y")
#turns dates into DATES in r
fidmeantemp<-ddply(fidy1v3,.(Date),summarise,mean_temp=mean(Temp,na.rm=T))
#creates mean temp file
fidmintemp<-ddply(fidy1v3,.(Date),summarise,min_temp=min(Temp,na.rm=T))
#creates min temp file
fidmaxtemp<-ddply(fidy1v3,.(Date),summarise,max_temp=max(Temp,na.rm=T))
#creates max temp file
fidmedtemp<-ddply(fidy1v3,.(Date),summarise,med_temp=median(Temp,na.rm=T))
#creates med temp file
ggplot(data=fidmedtemp, aes(Date, med_temp, group=1))+geom_line(color="purple",size=1.5)+geom_abline(intercept=12.5, slope=0,color="red", size=2)+scale_x_date(breaks="1 month", minor_breaks="1 week",labels=date_format("%B %Y"))+theme(axis.text.x=element_text(angle=45, size=10, vjust=0.5))

#plots median temps
ggplot(data=fidmintemp, aes(Date, min_temp, group=1))+geom_line(color="purple",size=1.5)+geom_abline(intercept=12.5, slope=0,color="red", size=2)+scale_x_date(breaks="1 month", minor_breaks="1 week",labels=date_format("%B %Y"))+theme(axis.text.x=element_text(angle=45, size=10, vjust=0.5))

#plots min temps
ggplot()+geom_line(data=fidmeantemp, aes(Date,mean_temp, group=1), color="Purple",size=1)+geom_line(data=fidmintemp, aes(Date,min_temp,group=1),color="Blue",size=1)+geom_line(data=fidmaxtemp,aes(Date,max_temp,group=1),color="Red",size=1)+geom_abline(intercept=12.5, slope=0,color="Red", size=0.5)+labs(x="Date",y="Min|Max|Mean Temperatures(C)")+scale_x_date(breaks="1 month", minor_breaks="1 week",labels=date_format("%B %Y"))+theme(axis.text.x=element_text(angle=45, size=10, vjust=0.5))

#plots all temps