3 31 2015 EZNA with Oyster Bay oysters Pt. 2

Today started using the new EZNA 200 reaction kit we received yesterday. Before beginning I added Isopropanol to the HBC buffer and 100% EtOH to one bottle of DNA Wash buffer. Then I continued the procedure as normal. These samples are related to the samples I extracted yesterday.

Before using the EZNA Kit I dissected out whole body tissue from 24 seed oysters into the homogenization tube. The oysters are labeled SN 7-30. 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 SN 

3/31/15 JH 2 of 2

3 30 2015 EZNA with Oyster Bay

Last week I started isolating DNA from seed oyster from Dabob and Fidalgo. Yesterday the new kit didn't come in until later so I started with the remaining six reactions from the previous kit. I got busy and forgot to post about it so here it is. The samples are labelled SN 1-6.

Before using the EZNA Kit I dissected out whole body tissue from 6 seed oysters into the homogenization tube. The oysters are labeled SN 1-6. 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 
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 
25. Discarded flowthrough
26. Added 700 ul DNA Wash Buffer
27. Centrifuged at 10,000 g for 1 minute 
28. Discarded flowthrough
29. Repeated 26-28
30. Centrifuged Empty column for 2 minutes at 10,000 g 
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 
1. Internal lid in centrifuge failed which resulted in tube caps being sheared off. 
2. Pipetted sample in collection tube to new tube
3. Moved the spin columns to the new tubes
35. Repeated as normal 32-34. 
1. Under calculated amount of Elution buffer needed to be preheated. 
2. SN 4, 5, and 6 had cold Elution buffer added instead
36. Stored DNA at -20 C

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

3 24 2015 EZNA Seed Isolation Gel Run

I ran a subset of the samples from yesterday. The samples were HL 1-6. The DNA looks very degraded still. There were only two of the 6 that I would consider usuable, 2 were questionable, and 2 were fully degraded. Moving forward, it looks like the EZNA kit can salvage usuable samples from some of the seed but not all of them. This is pretty similar to the Qiagen 96 Well Plate kit in this instance. Since we've already ordered the 200 reaction kit for the EZNA and I've processed 20 dabob seed and am processing another 20 Fidalgo seed today it seems only reasonable to continue using this kit.

Gel Protocol:

75 ml Low TAE with 0.6 g Agarose and 7.5 ul EtBr.

Run at 120 V for 55 minutes.

10 ul Ladder, 20 ul sample mixed with 3 ul loading dye.

Gel Layout:

Well	1	2	3	4	5	6	7	8
Sample	Ladder	HL 1	HL 2	HL 3	HL 4	HL 5	HL 6	Ladder

Gel with Dabob Seed DNA Isolation

Close up of High Molecular Weight Material. 

3 23 2015 EZNA DNA Isolation with Seed Oysters

Due to the success of the EZNA kit last week with frozen tissue from September 2014, We have decided to begin extracting DNA from seed oysters to develop high quality libraries for sequencing. Today I processed 20 Dabob Bay seed oysters from August 2013 with the EZNA kit. The process is basically identical to last weeks except I used the refridgerated centrifuge for a few steps because it could spin more samples than my desktop centrifuge.  

Before using the EZNA Kit I dissected out whole body tissue from 20 seed oysters into the homogenization tube. The oysters are labeled HL1-20. 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

Tomorrow I will run the DNA out on a gel to check for quality. 

3 20 2015 Aster Model

Aster Summary

Project

In an effort to produce an analysis of our reproduction and mortality data to define differences between populations and derive statistical inferences about local adaptation. One such method is to use the aster model which unlike linear and general linear models uses compounding effects of mortality on reproduction to derive information about fitness. I have investigated the use of the aster model to determine if it is right for the data we have.

Aster Background

The aster model was developed to investigate fitness which is typically difficult to quantify and create a statistical model that can account for compounding effects of fitness components. The two most important fitness components in the aster model is survival and reproduction. Survival of an individual directly impacts its future ability to reproduce which equals overall fitness of an individual. Most systems such as linear (LM) and general linear models (GLM) only investigate either mortality or reproduction which cannot statistically support any conclusions about fitness due to the interdependence of the two. Aster models were originally used to model the fitness of a prairie grass that existed in remnant populations. The aster model has been used in many other species to analyze differences in fitness explored in common garden and reciprocal transplant experiments. One arabidopsis species was used in a reciprocal transplant experiment in Europe. The researchers found a statistically significant difference in the transplanted populations with local populations being more fit than the introduced populations. It is for these reasons that I want to use the aster model with my data as it could potentially produce viable information about local adaptation inOstrea lurida.

What Oly Data Works with Aster

The data we've collected from the previous year about the three reciprocally transplanted populations in Puget Sound, WA has all the necessary parts to function with the aster package. The aster package requires three semi-dependent sets of data. First it needs mortality information for use with survival analysis. Next it requires some binary measure of reproductive activity. Finally it needs continuous data for reproduction such as number of seeds produced. We have mortality information for the year which is already in the binary data format. We have brooding female numbers for the year as well with a little bit of manipulation can be made into binary data. We also have counts of larvae which works as continuous data about reproduction.

What Does Not Work with Aster

The aster model was developed to analyze multiyear data sets with annual variation for the data set. Since our data is only for a single year, the variation in the data is too limited for use with the aster model from what I can tell.

Decision

At this time I don't believe that the aster package can be used to a good effect to determine relative fitness of the three populations at the sites. I'll continue working with the aster material if I can figure out how to make it work for single year data.

You can see my R script here.

3 18 2015 EZNA Gel Run

Today I ran the gel with the EZNA isolated DNA from frozen samples which I did yesterday. You can read the blogpost here. The sample quality looked much better than with the DNAzol or the Qiagen kit. It's still heavily degraded but there appears to be high molecular weight material in the isolation.

The gel was made with 75 ml Low TAE and 0.65 g Agarose. I also added 7.5 ul EtBr for staining.

Gel Layout

Well	1	2	3	4	5	6	7	8
Sample	Ladder	1H13-16 9	1H13-16 10	1H13-16 11	1H13-16 12	Ladder	Empty	Empty

It looks like the Omega kit does a really good job of isolating high molecular weight DNA from the frozen samples. This kit still has 46 reactions left in it. The entire protocol with 4 oysters took about 3 hours yesterday. I'm assuming with 10-20 oysters it will probably increase to 4-5 hours. I can isolate 10-20 samples at a time to keep the pace reasonable and still get good results if we want to pursue this course of action.