07/13/17

Through the belly of the beast: Drive through BC wildfire explained

Blog post by Jack Woods; video and images by Sally Aitken

Sally Aitken and I were at our cabin in the west Chilcotin near Tatla Lake for a week prior to the lightening storm that ignited so many fires in the east Chilcotin and the corridor from Cache Creek to Quesnel on July 7th. Along with others in the area of our cabin, we monitored the status of the fires and were concerned about being able to leave should highway 20 (the only realistic exit route) close. Another fire in the Kleena Kleene area to the northwest of us was threatening to close highway 20 west bound to Bella Coola. On July 8th, highway 20 was closed in both directions due to the Hanceville fire and power to the west Chilcotin went out due the Hanceville fire damaging power lines. We packed for a quick exit on July 9th and, as there were no other options, went for a mountain bike ride. When we returned from our ride in the late afternoon we learned that Highway 20 was open eastbound as an escape route, but was still closed westbound. We made a hasty exit.

At about 8:00 pm we passed through Alexis Creek heading towards the fire. The winds were from the west blowing smoke away from us, so we had good views of the Hanceville fire on the hills to both the north and south of Highway 20. Fire crews and heavy equipment were working to put in fire guards to protect homes in the Anaham Reserve. As we drove further east, the fire came closer to the road and we drove through areas that had already been largely burned, with residual spot fires that were still active. Lee’s Corner gas station and restaurant was burned down. Proceeding further east from Lee’s corner, we drove into the active fire area, with burning on both sides of the road. This is where some of the video begins.

Visualize for a moment a massive fire that is many square kilometers in size working it’s way through a somewhat open conifer forest and grasslands that are tinder dry. A westerly wind is pushing a plume of smoke to the east. The plume is thin on the west edge and rises like a massive wedge as you move to the east. We were driving from the thin edge of the smoke wedge into the thick edge. It got steadily darker.

Forests burned by ground fire

Burned grasslands

The fire was burning to the road edge on both sides and there was some crowning visible (for non-foresters, crowning is when the fire burns explosively through the foliage in the upper part of a tree). As it got darker the flames appeared more orange. Proceeding further east the darkness became complete and we forgot that the sun had not yet set. We saw periodic crowning, burning stems, and ground-level fires of varying intensity burning erratically on both sides of the road. Whole stands of trees in some spots had turned to red hot, glowing columns of orange light. Driving through the smoke was like driving through a blizzard. The road was only visible with the headlights on low beam and we could only see from 10 to 30 meters ahead. Several times intense fire-generated winds swept across and totally obscured visibility. Sparks and embers were common.

Screenshots from video. View video here: https://www.youtube.com/watch?v=860OpMy8kYc&t=2s

We proceed east through the dark with the fire on either side for about 20 km (from just after the old mill at the top of the Hanceville hill to about Harper Lake). At Harper lake the smoke began to clear somewhat and the fire was further from the road. It was still very dark, but we were able to use high beam and see further ahead. Ominous glows appeared here and there through the smoke and we were very concerned that we might drive into still more of the intense part of the fire. Finally a brighter orange glow appeared ahead and on both sides. Our first reaction was fear that the worst of the fire still lay in front of us. We were relieved, however, when we realized that this new glow was daylight and we were finally emerging from the fire.

The view back when we emerged from the black cloud of smoke. Heavy equipment was building fire breaks on this hillside.

 

Why didn’t we turn back early on? The trees in this area are not large (few over 20 m in height) and the sides of the road are cleared back some 20 or more meters. The chance of the road being blocked by a fallen tree was very small. We knew that the darkness was due to the thickening of the smoke wedge above us, and although the fire in the darkness looked frightening, it was in reality no worse than what you see in the early part of the video. We monitored the external temperature as we drove. It was about 22C as we entered the fire and increased to about 25 to 27 degrees. A couple of times it spiked to about 35 degrees and we considered turning around, but each time it quickly returned to the mid to high 20’s. As ominous as it all seemed, we were not in serious danger as long as we drove carefully and the outside temperature stayed reasonably low. So, we pushed forward and after what began to seem like an interminable amount of time, emerged on the east side of the fire. Our dog slept in the back, completely unaware of anything but the bag of treats in the glove box. Similar to a small group of cows we saw grazing in a small field, apparently oblivious to the fire all around them.

The video of a small part of our drive is here. Unfortunately, perhaps, Sally was so focused on the fire she lost focus on capturing more of it on video. Priorities.

There are many victims of this fire and the many other fires burning in central BC. This is the real issue. Please consider a donation to the Red Cross or another organization working to help people through this. In addition to our personal donations, we will also donate any funds we receive through commercial use of this video to the Red Cross relief effort.  https://donate.redcross.ca/ea-action/action?ea.client.id=1951&ea.campaign.id=74010&_ga=2.234106270.1847245054.1499898622-97910928.1499898622

 

 

 

 

03/7/17

CoAdapTree in pictures

Here are some pictures showing the progress on our new project, CoAdapTree.

1. Western larch for transcriptome analysis

 

 

 

 

 

 

2. Lodgepole pine for inoculation pilot

3. Inoculation pilot

4. Lodgepole pine controlled crosses RxR and SxS

5. Pine : 40 seedlots

6. Collection of BC lodgepole pine (and some AB Jack pine): 40 seedlots

7. Sowing 4000 pine seedlings

8. Jack pine for transcriptome analysis

 

 

9. Jack pine RNA extraction

 

 

5.7

01/12/17

We do other things too: extracting DNA from cambium

This post is about labwork. Anyone reading our blog might have the idea that being in the Aitken lab is much like being Indiana Jones (or possibly Tarzan) – and sure, we are a good-looking bunch of rugged adventurers. But the truth is that although most lab pictures show our feats of courage ascending massive Sitka spruce, or wandering the wilds of Alaska, we spend a lot of time in the lab.

Joane Elleouet and I tried to extract DNA from dried Sitka spruce cambium for six months or so last year, on and off. We went through a number of protocols, and although we eventually modified one of them enough that it mostly worked, we were amazed at the lack of guidance online for cambium extractions. Surely someone had done this before! Nope. We thought that the world wide web had our back, and then it didn’t.

This is the wetlab, the room that hears the most cursing and cheering. We (mostly Dragana) recently cleaned, which is why it looks so good.

What you’ll find below is an annotated version of a CTAB DNA extraction protocol we got from Kristin Nurkowski (who got it from Marco Todesco at the Rieseberg Lab),  which we have modified for cambial tissue. It’s not perfect, but if you’re having trouble getting DNA out of cambium it may be your best bet. Probably some more improvements can be made. Note also that the suggestions we offer are just what one or both of us thought worked best for our own samples as we extracted them: we haven’t tested them with other batches of samples, and often we haven’t tested each alteration to the original protocol in isolation. But despite our recalcitrant tissue, we managed to scrape a 60-90% success rate, depending on the exact methods and samples (which is actually pretty good). Hopefully you can do as well or better.

When not in the lab or the field, this is what we do: Joane, Ian, Jon, and Pia being productive.

Unfortunately, for those of you who are just reading this for fun, recess is over once you cross the dashed line below.

————————————— Beginning of the protocol —————————————

The protocol has been through a number of modifications, but originally it was from Zeng et al. 2002, Acta Botanica Sinica 44; 694-697. There are a few other papers published that have other methods for extracting DNA from cambium (preserved in different ways) that you could look for in case of difficulty.

PART I: Preparing the buffers

CTAB-free Buffer (100 mL total volume):

20 mL 1M Tris-HCl stock, pH 8.0
10 mL 0.5 EDTA stock, pH 8.0
5 mL 5M NaCl stock
65 mL PCR-clean H2O
Autoclave

3% CTAB Buffer (100 mL total volume):

10 mL Tris-HCl stock
5 mL EDTA stock
30 mL NaCl stock
3 g CTAB
1 g PVP
55 mL PCR-clean H2O
Do not autoclave, the PVP will be destroyed.

High Salt TE (100 mL total volume):

20 mL NaCl stock
80 mL TE buffer
Autoclave

TE Buffer (100 mL total volume):

1 mL Tris-HCl stock, pH 8.0
200 uL EDTA stock, pH 8.0
98.8 mL PCR-clean H2O
Autoclave

PART II: Tissue collection

We used discs of cambium and phloem (since it’s difficult to get just cambium – it’s all stuck together in the inner bark) obtained using a leather punch from the base of Sitka spruce trees and dried in silica gel. We shaved around 20-30 mg (usually 22ish) of tissue from the inside (wood side) of each disc. That’s the region that should contain the (supposedly) DNA-rich cambial tissue.

Our samples weren’t perfect to begin with. Mine were collected in late July on Vancouver Island, after the peak of cambial activity in the spring. Collecting earlier might have improved the yield. Joane’s samples had been dried in silica gel for a year, and it’s possible they became less tractable with time.

Put your tissue into a 2 mL tube with one steel ball bearing. It is really important to weigh tissue into a 2 mL tube because the ball bearing can get stuck in the end of the 1.5 mL tube.

PART III: Grinding

1. Place your samples in a mixer mill block, put it in a Styrofoam box, and pour liquid nitrogen over it (wear insulated gloves!).
2. Assemble the block and fit it into the mixer mill.
3. Grind samples at 30 Hz for 1 minute.
4. Quickly disassemble the block and freeze each part again.
5. Reassemble the block, flipping it 180◦ to ensure even grinding, and grind again at 30 Hz for 1 minute. Usually, redistributing the samples within the block each time helps them grind evenly.
6. Disassemble the block and check each sample. It should look like a fine beige powder. If the sample is not ground properly, freeze everything and grind it again. For our samples, it took as many as ten repetitions of the grinding process.
7. After grinding, quickly add the prepared buffer and start the protocol.

PART IV: Protocol

8. Put some 3%CTAB to warm at 65◦F with loose cap
9. Add 1 ml of CTAB-free buffer (cold) + 6 μl β-mercaptoethanol (mix done beforehand in the fumehood with a bit extra)
10. Mix by inversion (shake each sample quickly), keep 10 minutes in the freezer. While waiting, put some 3% CTAB in the waterbath and some 2-propanol in the freezer
11. Spin at 10.000 g for 10 minutes in the centrifuge (longer may be required, depending on your tissue)
12. Discard the supernatant under the fumehood in a trash beaker
13. (if you use particularly nasty tissue, you can repeat this step)
14. Add 500 μl of pre-warmed 3% CTAB + 5 μl of β-mercaptoethanol in the fumehood
15. Incubate at 65°C for 60 minutes
16. Let the tubes cool down a little bit
17. Add 500 μl of chloroform-isoamylalcohol 24:1 in the fumehood. SUPER CORROSIVE!
18. Vortex
19. Spin at full speed for 15 minutes. Meanwhile, label new 1.5mL tubes
20. Move the aqueous phase to the new tubes (1.5mL) under the fumehood. This must be done exactly right – it’s probably the most important step. The aqueous phase is the liquid above the solid phase in the centre of the tube. Don’t take all the liquid, and don’t disturb the part that’s within a couple mm of the solid-liquid interface. Take around 320 uL from the top surface of the aqueous phase slowly and carefully. Too much will give you an impure sample. Too little will decrease the yield of DNA.
21. Add 250 μl of NaCl 5M, mix fast
22. Add 500 μl of cold isopropanol (2-propanol), mix (have a tube ready in the freezer)
23. Leave at least 20 minutes or overnight at -20°C. You can take a break here if you want!
24. Spin at max speed for 15 minutes
25. Wash pellet in fumehood with 1 ml of cold 80% ethanol (prepare the mix from 100% and PCR-clean water)
26. Spin at max speed for 5 minutes
27. Repeat wash (no need to be under the fumehood anymore)
28. Dry pellet (by putting the tubes in the heatblock at 25 to 37 degrees)

Depending on your tissue, you may need to include the following seven optional steps (labelled 29-35). Otherwise proceed directly to step 36. We found that omitting the optional steps often led to severe RNA contamination. The Nanodrop would say we had lots of very pure DNA, but the Qubit (which is much more reliable) would say we had almost none.

29. Resuspend in 400 μl of high salt TE + 2 μl of RNAse A (sometimes more RNAse is needed)
30. Incubate at 37°C for one hour, mixing occasionally until the pellet dissolves. Sometimes this is a LOT of mixing.
31. Add 800 μl of cold 100% ethanol
32. Spin at max speed for 15 minutes
33. Wash pellet with 1 ml of cold 80% ethanol. If the pellet is invisible, it doesn’t mean the sample failed. Just continue, leaving a bit of liquid at the bottom of the tube with every wash, and be very careful not to touch the part of the tube where the pellet should be when you suck out the liquid to dry the pellet.
34. Spin at max speed for 5 minutes
35. Repeat wash

36. Dry pellet and resuspend in 50 uL elution buffer or TE. Use less if you need higher concentrations. The yield of this procedure was often in the range of 250 – 1250 ng when the optional steps (29-35) were included, and more if they weren’t.
37. Quantification and quality control: we used a Nanodrop and a Qubit. The Nanodrop gives (sometimes wildly) inaccurate DNA concentration readings but it can test for purity. The Qubit gives accurate concentrations, but doesn’t tell you what might be in the sample along with the DNA.

Hazards

It is important to note the two most hazardous reagents used in the protocol. In addition to being stinky, the B-mercaptoethanol is also toxic. Any supernatant or plastic item contaminated by it must remain in the fume hood and becomes hazardous waste. Do not breathe it in or get it on your skin. The chloroform is also toxic and can burn your skin and lungs. Keep the lid on and it in the fumehood! Any plasticware contaminated with chloroform needs to remain in the fumehood until dry, and then it can be thrown out with regular trash. Liquid waste needs to be stored and disposed of according to your local regulations (but not down the drain!).

Advice

The biggest problems with this protocol were:

• A mystery contaminant. There seems to be something in Sitka cambium or phloem that gives low 260/230 ratios on the Nanodrop (i.e. a bad quality score), if it isn’t properly removed. Sometimes it removes well, other times not.
• RNA contamination. We sometimes had lots of RNA in the samples if we skipped steps 29-35. If this isn’t a problem for you, omit them. If it is, include them – but the whole protocol gets more finicky when you do. Getting rid of the RNA while keeping enough DNA to work with (in our case for GBS and sequence capture and then next-gen sequencing) wasn’t always easy.
• Low yields. Sometimes we just didn’t get much DNA. The good news is, this often seemed to be protocol-dependent rather than sample-dependent. When a sample didn’t work out, trying the same sample again often solved the problem.

Some suggestions for troubleshooting:

• Try changing the amount of supernatant you take in step 20. Take more if you have no DNA. Take less if you have a contamination problem.
• Try starting with different amounts of tissue. Use more if you have no DNA. Use less if you have a contamination problem.
• Be careful washing the pellet: don’t lose all or part of it! Maybe spinning at colder temperatures (4°C? 10°C?) would help.
• Consider using RNAse A if you still have a contamination problem after including the optional steps. Consider eliminating the RNAse A steps if you have no DNA.
• Try using your newest tissue, collected whenever cambium is active near you (probably spring).

Final note:

All the extra cautions and advice I added to the protocol might make it seem a bit daunting. But remember, it actually works! The protocol isn’t particularly difficult, and maybe it’s less finicky than my comments make it seem — and after a few repetitions you should be churning out extracted samples no problem. So good luck!

08/23/16

Aitken and collaborators talking to the Verge about climate change and forests

Whitebark pines are majestic trees with a whitish, often wind-curled trunk that grow up high in the Rocky and Sierra Mountains, in the Western US. They’re icons of Yellowstone National Park, where they provide high-calorie seeds for many animals, including grizzly bears that eat the seeds before hibernating. Some whitebark pines manage to live for a thousand years, but many of them are now dying.

Source: Uprooted: how climate change may kick off an artificial migration of trees | The Verge