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. 

3 17 2015 DNA Isolation using E.Z.N.A Mollusc DNA Kit

Since last week I found that fresh samples produced the High Molecular Weight DNA I wanted. I've been trying troubleshooting the frozen samples. Today we received a kit from Omega Bioscience that is supposedly optimized for Mollusc DNA which can be troublesome. Today I used 4 samples from the September 2014 collection at Oyster Bay to test this kit for frozen tissues.

The samples were 91912014 1H13-16 9/10/11/12.

First the kit assumes that the samples are stored or processed with liquid nitrogen as it asks you to used a mortar and pestle to grind the tissue into powder. Our samples are not preserved this way so I modified the first step to work with our samples.

Before beginning the procedure I also mixed all the reagents necessary for the kit. I added Absolute Ethanol to the DNA wash and pure Isopropyl alcohol to the HBC buffer in the quantities required. These bottles have been labelled accordingly for future reference.

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
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 12 2015 DNAzol with Fresh Tissue Pt. 2

Today I ran a gel using the freshly isolated DNA from yesterday. You can see the post about it here. The gel protocol is as follows.

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

Loaded the wells with 10 ul 100 bp ladder and 15 ul DNA loading solution (Mix of 15 ul DNA and 3 ul loading dye).

Well	1	2	3	4	5	6	7	8	9	10	11	12
Technique		Centrifuged				Spooled
Sample	Ladder	Test Oly 1	Test Oly 2	Test Oly 3	Test Oly 4	Test Oly 1	Test Oly 2	Test Oly 3	Test Oly 4	Ladder	Empty	Empty

Gel with Fresh Tissue Isolation

I have no idea where the DNA when. I saw a ton of it yesterday when I was extracting the DNA. I'm wondering if I missed it. When I was pulling the sample to load as it may have sank to the bottom of the tube and I collected off the top. On the bright side you can see two bands of HQ High molecular weight DNA. in the Centrifuged set. I will run another gel tomorrow to see if I can get better samples to appear on here. 

This makes me wonder if there is something wrong with the freezer tissue that has degraded the High molecular weight stuff. I talked to Andy yesterday and he told me all of his sablefish samples were stored in EtOH to avoid degradation. Either way this is up for consideration.

**Update 3/13/2015**
I ran the same protocol again this morning except this time I mixed the samples very well before pulling a sample from them. After running the gel, it looks almost identical except sample #1 looks better in the second sample. I'm not sure what happened with this protocol but supposedly that single band is confirmation of high quality high molecular weight DNA.

3 11 2015 DNAzol Isolation with Fresh Tissue

So we've been having trouble with our tissues from previous samplings. Since they have all been preserved dry in -20 conditions for months to over a year we decided to attempt an extraction on Olympia oysters fresh from a hatchery. I collected 4 oysters from a South Sound population from the Ken Chew Hatchery thanks to Puget Sound Restoration Fund and Ryan Crim. I performed the first half of the DNAzol isolation tonight with tissues that I dissected out of living animals. Samples ranged from 30-70 mg in size which is close enough to the proper DNAzol range. Like the last time I did a DNAzol extraction, I split the samples into spooled and remnants. Surprisingly, the DNA spooled very very well. There was a clear difference between how these samples behaved during the extraction process and the previous samples. All reagents and techniques are identical to the previous extraction so no difference should be observed unless there's something to fresh extractions.

Protocol:

1. Dissected out grain of rice size piece of mantle tissue from each oyster and placed in collection tube. 
2. Added 1 ml DNAzol reagent to tube. 
3. Homogenized using pestle for 45 seconds
4. Incubated at room temp for 10 minutes
5. Centrifuged sample at 10,000 g for 10 minutes
6. Extracted supernant and placed into a fresh tube
1. Large goopy looking clumps in supernant that had clearly not compacted during centrifugation were present. 
7. Added 500 ul 100% EtOH to supernant
1. Instantly produced large amount of bright white material
2. After inversion mixing, large goopy strings of precipitate formed on the bottom
8. Spooled goopy material on the bottom of the tube and placed into new tube
9. Centrifuged remaining supernant at 5,000 g for 5 minutes.
1. No pellet formed in these tubes. 
2. Slight film on tube walls.
10. Added 1 ml 75% EtOH to tubes.
1. In the original tubes nothing happened
2. In the spool tubes bright white strings of material appeared and solution clouded up
11. Incubated for 1 minute at room temp.
12. Centrifuged tubes at 1,000 g for 2 minutes
1. spooled tubes had large amount of material still in suspension
2. Recentrifuged these tubes at 10,000 g for 2 minutes
3. film and pellet formed from spooled tubes
13. Removed EtOH as best I could
1. Due to strings of material I couldn't remove all of it
14. Eluted samples with 300 ul of Nanopure water. 

All in all, these samples behaved totally different. Previous samples would not spool and remained in supernant which formed a solid pellet. These samples spooled very well, leaving little in the supernant and formed a film and loose pellet of material. Adding Ethanol made DNA precipitate visibly unlike before. 

I will run these samples on a gel tomorrow but I get the feeling they are going to have high quality DNA in them. If this works, future samples should be collected and isolated immediately or within a short time frame. 

3 2 2015 DNAzol Extraction Attempt 2

A little over a week ago I attempted to isolate DNA using DNAzol and DNEasy to collect high molecular weight DNA. You can see the results here. I originally thought we had collected HMW DNA as the smears were mostly above the 1000 bp portion of the ladder. After discussing the results with Brent and Steven, it appears the HMW DNA is not large enough and should be a tightly condensed band at the top. Today I am extracting more DNA using DNAzol to try to extract only the HMW DNA using a spooling technique. The samples I'm using are the same ones from before since I had plenty of left over tissue (9-19-2014 1N9-12 18,19,20,21). To compare the spooling technique to the centrifuge technique, I spooled as much DNA as I could and transferred it to a clean second tube. Then centrifuged the original sample tube to collect a pellet. If I successfully collected the HMW DNA with the spooling it shouldn't appear in the centrifuged sample. If I didn't collected it, then the spooled sample should have little to no DNA. The rest of the protocol is as follows. I will run a gel on the samples this afternoon to check the sample quality.

DNA Isolation:

1. Subsampled very small portion of tissue from original tubes and placed into homogenization tube. 
2. Added 1 ml DNAzol to each tube
3. Homogenized with 10 strokes of the pestle.
4. Incubated at room temp for 10 minutes.
5. Centrifuged at 10,000 g for 10 minutes.
6. Transferred supernant to new tube.
7. Added 0.5 ml 100% EtOH. 
1. Originally saw a milky precipitate form but then disappear after mixing through inversion.
8. Mixed through inversion (8 inversions)
9. Incubated at room temp for 3 minutes. 
10. Stuck a clean 200 ul pipette tip into solution and gently swirled solution around tip to spool DNA. Transferred to new labelled 1.5 ml tube and gently slid the tip across the inner surface of the tube.
1. No visible spooled DNA. I repeated spooling multiple times to collect as much as possible but saw no visible DNA.
11. Added 1 ml 75% EtOH to spooled DNA tube.
12. Centrifuged original tube 5,000 g for 5 minutes. 
13. Removed supernant from centrifuged tube.
14. Added 1 ml 75% EtOH to Centrifuged DNA tube.
15. Centrifuged all tubes at 1,000 g for 2 minutes to collect DNA in the bottom. 
16. Carefully removed all remaining EtOH from all tubes. 
17. Added 300 ul Nanopure water to each tube.
18. Mixed through pipetting. 
19. Stored at 4C until I can run the gel. 

**UPDATE**

I completed the gel run this evening for quality checking. It appears the spooling failed but upon examination of the centrifuged samples that there is a load of HMW DNA remaining in the well that did not travel through the gel. So the DNAzol works with the centrifuge extraction. 

0.8% Agarose Gel with Low TAE and 5 ul of EtBr. Ran gel at 120 v for 40 minutes.

Gel Layout

Well	1	2	3	4	5	6	7	8	9	10	11	12
Technique		Spooled				Centrifuged
Sample	Ladder	1N9-12 18	1N9-12 19	1N9-12 20	1N9-12 21	1N9-12 18	1N9-12 19	1N9-12 20	1N9-12 21	Ladder	Empty	Empty

Full Gel

High Molecular Weight DNA remaining in Well.