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. 

2 18 2015 96 Well Plate Extract Gel Run

Today I ran half of the 96 well plate extraction (part 1 and part 2) on a 1.3% agarose gel to determine the quality of the DNA isolated. Sadly the DNA is heavily degraded and there appears to be no high molecular weight band. To fit all of the samples, I did not add a ladder which would tell us exactly what the weight of the DNA is. Overall this is very disappointing. I'm going to run the other half tomorrow to see if maybe they are of better quality or may there was a loading issue with the samples.

Protocol for today:

Used a super sized gel mold and wells. So it took a pretty big volume.

Gel:
500 ml 1X Low TAE
6.5 g general purpose agarose


Microwave solution for 3 minutes at a time until boiling.
Visually inspect for dense materials (apparently I didn't do well enough as one corner of the gel was higher density than the others.

Placed two rows of 24 well combs in gel mold.
Slowly poured gel into one corner of the mold.

Allowed get to set for 30 minutes.
Once gel was firm I placed it in the electrophoresis chamber.
I filled the chamber with 1 X Low TAE solution until it was even with the gels top surface.

Pipetted 30 ul of Low TAE into each well. Then loaded wells with DNA using the 12 channel pipette in a left to right, top to bottom pattern.

Ran gel for 10 minutes at 120 v.
Paused gel and added 1 ul 5X loading dye to the left most well on both rows.
Ran gel for another 10 minutes at 120 v.
Paused gel and added 1X Low TAE until gel was thoroughly covered.

Ran gel for 1.5 hours until dye had moved appropriately far down the lane.

Placed gel in EtBr bath made 1 L water and 1 ml EtBr.
Soaked gel for 30 minutes.

Imaged on the translinker.

Samples organized:

Well

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

Top Row

1N1-4 1

1N1-4 2

1N1-4 3

1N1-4 4

1N1-4 5

1N1-4 6

1N1-4 7

1N1-4 8

1N1-4 11

1N1-4 10

1H1-4 1

1H1-4 2

1H1-4 3

1H1-4 4

1H1-4 5

1H1-4 6

1H1-4 7

1H1-4 8

1H1-4 9

1H1-4 10

1S1-4 1

1S1-4 2

1S1-4 3

1S1-4 4

Bottom Row

1S1-4 5

1S1-4 6

1S1-4 7

1S1-4 8

1S1-4 9

1S1-4 10

2N1-4 1

2N1-4 2

2N1-4 3

2N1-4 4

2N1-4 5

2N1-4 6

2N1-4 7

2N1-4 8

2N1-4 9

2N1-4 10

2H1-4 1

2H1-4 2

2H1-4 3

2H1-4 4

2H1-4 5

2H1-4 6

2H1-4 7

2H1-4 8

Full Gel

Top Row Samples A1-12, B1-12

Bottom Row Samples C1-12, D1-12

2 17 2015 Bioanalyzer Results for BS Libraries

Today I was able to look at the bioanalyzer data that Sam sent me. It looks like most of the samples failed. A couple of the samples had very small peaks between 300-500 bp but they were basically negligible. You can also see some faint smearing in those regions in the gel view but again there was so little material that it probably not possible to retrieve any useful data from them.

Gel View of the Bioanalyzer data. 

Electropharogram View, minimal peaks seen

Electropharogram View of the 3 samples with noticeable peaks. 

2 13 2015 oyster bay sample move

Oyster Bay Wa
1030 am to 1 pm
Mid 50s foggy.

Moved the samples today from the calm fresh dock to the taylor mussel raft. 

Checked the samples quickly for mortality. Low to no mortality in each container. All animal look good and healthy. Fidalgo animals look much larger than the other two pops. They are also super dense. They feel like they weigh 50-75 grams. Compared to oyster and dabob which feel between 30-50 grams. 

Due to time constraints for the taylor boat, I was not able to image any populations for size. I pulled the logger data on both tags.  Samples were hung on the taylor rafts with bright orange tags with contact info.

Finally the new dock at crab fresh will be completed by the end of march. I plan to move the samples back from the taylor raft in mid april.

2 12 2015 DNA Isolation Part 2

As with yesterday's post, I'm finishing up the 96 Well Plate Qiagen DNEasy extraction kit. Today I worked in the MERLab because of the proximity to the centrifuge that can fit the kit plates. I brought all the reagents, pipetters, pipette tips, and gloves. I ended up borrowing a graduate cylinder but I cleaned it thoroughly before and after use. A couple things to note, I lost some of the lysate from the samples as a couple of the caps were not as secure as I thought were and popped open during the first shaking step. Luckily these mostly came from the first column which as you can tell by the plate set up only affected one or two samples from each population sampled. Also on the elution step I repeated it twice because on the first run through, Row A received half as much buffer as intended. When I repeated the elution I over filled Row A and now there is about 500 ul of isolate instead of 400 ul. I will run a gel on the samples next week to check quality. Hopefully Row A is not extremely diluted. Though I can probably concentrate it without too much trouble. Also I used the sample channel reservoir for every solution. Between solutions I rinsed with water and did a final rinse with DI Water. I wiped it dry with a paper towel.

Today's Procedure:

1. After incubating roughly 24 hours at 56 C samples were sealed and shaken (lost some lysate due to poor seals on a few caps).
2. Centrifuge at 5700 rpm for 20 second.
3. Uncapped/Added 410 ul premade Buffer AL/EtOH solution.
4. Capped with new caps. Shook for 20s.
5. Centrifuge at 5700 rpm for 20 second.
6. Placed a DNeasy 96 well column plate on the previously used S Block. 
7. Attempted to remove 600 ul of lysate from each sample
1. Some had more some had less due to volume of tissue and loss of lysate. Most sample tubes appeared visually to be at roughly the same volume.
8. Sealed plate with Airpore sheet.
9. Centrifuged at 5700 rpm for 11 minutes.
10. Decanted solute from S Block. Wiped with Paper towel. 
11. Removed Airpore tape. 
12. Added 500 ul Buffer AW1 to each sample
13. Seal with Airpore tape.
14. Centrifugee at 5700 rpm for 6 minutes. 
15. Removed Airpore tape. 
16. Added 510 ul Buffer AW2 to each sample. 
17. Centrifuged at 5700 rpm for 16 minutes. (Used no airpore tape per instructions)
18. Moved DNeasy 96 well column plate to Elution Microtubes plate. 
19. Added 200 ul Buffer AE to each sample (Row A got ~100 ul)
20. Sealed with Airpore tape. 
21. Incubated for 1 minute at room temperature. 
22. Centrifuged at 5700 rpm for 3 minutes. 
23. Removed Airpore Tape. 
24. Added 200 ul Buffer AE to each sample (Row A got 400 ul)
25. Sealed with Airpore Tape
26. Incubated for 1 minute at room temperature. 
27. Centrifuged at 5700 rpm for 3 minutes. 
28. Removed DNeasy column block from Elution Microtubes plate. 
29. Sealed Microtubes with rubber tops flipping which which side the openning tab was on for every column. 
30. Labelled sample with "O.lurida sample processed 2/12/2015. 09/14 Oyster, 10/14 Fidalgo Manchester. JH"
31. Stored in the 4 C in 213.