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 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 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 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 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