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.

2 20 2015 Test DNA Extraction Methods Part 2

Yesterday, after discussing the poor results from the 96 well plate extraction, I decided to directly compare the Qiagen DNEasy kit with the DNAzol method. You can see what I did in yesterday's blogpost. Today I completed the DNEasy extraction with only an overnight incubation instead of 24 hours due to concerns of dnases destroying genomic DNA during the incubation. I started the lysis incubation yesterday.

Today I performed the following protocol for DNEasy extraction.

1. Vortexed Samples to mix up lysed tissue
2. Allowed them to sit for 10 minutes while I made up a gel. 
3. Added 200 ul Buffer AL with EtOH from the 96 Well Kit
4. Added 200 ul 100% EtOH
1. This was a mistake as the 96 well kit premixes them and the singles kit does not. We ended up using a 75% EtOH, 25% Buffer AL Solution. 
5. Vortexed thoroughly
6. Pipetted into a column
7. Centrifuged at 6000 g for 1 minute
8. Discarded collection tube
9. Added 500 ul AW1 for wash and new collection tube
10. Centrifuge at 6000 g for 1 minute
11. Discarded collection tube
12. Added 500 ul AW2 for wash and new collection tube
13. Centrifuged at 10,000 g for 6 minutes
14. Discarded collection tube
15. Placed column in labelled 1.5 ml tube
16. Added 200 ul AE elution to column
17. Incubated 1 minute at room temp
18. Centrifuged at 6000 g for 1 minute
19. Repeated steps 16, 17, 18. 
20. Discarded column and stored at room temp. 

Following the completion of the DNeasy extraction I ran a gel with both sets of isolations. 

0.9% Gel:

50 ml 1X Low TAE

0.45 g Agarose

Microwaved gel for 4 minutes. Allowed to cool for 10 after thoroughly dissolved. Poured gel smoothly. Allowed to set for 30-45 minutes. 

Loaded wells with 

10 ul 100 bp Ladder

25 ul DNA with 2.5 ul Loading dye. 

Wells organized like:

Well	1	2	3	4	5	6	7	8	9	10	11	12
Technique		DNAzol				DNEasy
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	Empty	Ladder	Empty

Gel comparing DNAzol to DNEasy

First, There does appear to be intact High Molecular Weight DNA. Second, surprisingly the Qiagen produced a higher yield in the same sample. This makes me wonder if the 24 hour incubation allowed more DNases to chew through the DNA. If I do the plate extraction again, I will only allow for a 12-18 hour incubation period. Also I think the smaller tissue size helped immensely. It look like high molecular weight does vary between samples with less of it in two of the samples. Since all samples were processed the same way I'm not sure what this could mean in terms of sample quality and yield for sequencing. 

Moving forward, we should consider doing the 96 well extraction again with smaller tissue sizes, less incubation time, and possible the 75/25 mix of the AL solution.

2 19 2015 Test DNA Extraction Methods

Due to the low yields of high molecular weight DNA in the 96 Well Plate extraction and some advice from Brent and Steven, I'm now directly comparing DNEasy extractions with DNAzol. I selected 4 samples from the Oyster Bay September 2014 samples (1N9-12 18,19,20,21) to extract. I put equal amounts (visually pieces about the size of a grain of rice made of mantle and ctenidia) of each sample into two separate 1.5 ml tubes. One set of tubes is being processed using the DNEasy single sample kit with reagents from the 96 Well plate kit since both use the same reagents and the 96 Well plate kit is far fresher. The other set of tubes has been processed using the standard DNAzol technique.

The DNEasy kit takes an overnight incubation at 56 C so I filled the tubes with 180 ul ATL buffer and 20 ul Proteinase K. Vortexed to mix, centrifuged for 30 seconds, vortexed to resuspend and stuck into the incubator. These tissues will be processed tomorrow.

The DNAzol kit protocol went as follows.

1. Added 1 ml DNAzol to tissue tube. 
2. Homogenized using cleaned/previously used pestle with 5-10 strokes and grinding.
3. Incubated at room temp for 10 minutes. 
4. Centrifuged at 10,000 g for 10 minutes. 
5. Moved 700-900 ul supernatant to fresh tube.
6. Added 500 ul 100% EtOH (bottled opened 1/20/2015)
7. Mixed through inversion. 
8. Incubated for 3 minutes at room temp. 
9. Centrifuged at 5000 g for 5 minutes
10. Removed supernant
11. Washed with 1 ml 75% EtOH
12. Vortexed until pellet broke up. 
13. Centrifuged at 2000 g for 2 minutes. 
14. Removed supernatant.
15. Washed with 1 ml 75% EtOH
16. Vortexed until pellet broke up. 
17. Centrifuged at 2000 g for 3 minutes.
18. Carefully removed all supernatant. Used 1 ml pipetter to remove bulk and 200 ul pipetter to remove leftovers. 
19. Added 300 ul Nanopure water.
20. Mixed through pipetting. 

Lots of DNA created. DNA elution is almost milky in two of the samples. 

Will quantify tomorrow after I finish DNEasy extraction.