When not in the lab or the field, this is what we do: Joane, Ian, Jon, and Pia being productive.<\/figcaption><\/figure>\nUnfortunately, for those of you who are just reading this for fun, recess is over once you cross the dashed line below.<\/p>\n
————————————— Beginning of the protocol<\/strong>\u00a0—————————————<\/p>\nThe 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\u00a0in case of difficulty.<\/p>\n
PART I: Preparing the buffers<\/strong><\/p>\nCTAB-free Buffer (100 mL total volume):<\/p>\n
20 mL 1M Tris-HCl stock, pH 8.0
\n10 mL 0.5 EDTA stock, pH 8.0
\n5 mL 5M NaCl stock
\n65 mL PCR-clean H2O
\nAutoclave<\/p>\n
3% CTAB Buffer (100 mL total volume):<\/p>\n
10 mL Tris-HCl stock
\n5 mL EDTA stock
\n30 mL NaCl stock
\n3 g CTAB
\n1 g PVP
\n55 mL PCR-clean H2O
\nDo not autoclave, the PVP will be destroyed.<\/p>\n
High Salt TE (100 mL total volume):<\/p>\n
20 mL NaCl stock
\n80 mL TE buffer
\nAutoclave<\/p>\n
TE Buffer (100 mL total volume):<\/p>\n
1 mL Tris-HCl stock, pH 8.0
\n200 uL EDTA stock, pH 8.0
\n98.8 mL PCR-clean H2O
\nAutoclave<\/p>\n
PART II: Tissue collection<\/strong><\/p>\nWe used discs of cambium and phloem (since it\u2019s difficult to get just cambium \u2013 it\u2019s 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\u2019s the region that should contain the (supposedly) DNA-rich cambial tissue.<\/p>\n
Our samples weren\u2019t 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\u2019s samples had been dried in silica gel for a year, and it\u2019s possible they became less tractable with time.<\/p>\n
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.<\/p>\n
PART III: Grinding<\/strong><\/p>\n1. Place your samples in a mixer mill block, put it in a Styrofoam box, and pour liquid nitrogen over it (wear insulated gloves!).
\n2. Assemble the block and fit it into the mixer mill.
\n3. Grind samples at 30 Hz for 1 minute.
\n4. Quickly disassemble the block and freeze each part again.
\n5. Reassemble the block, flipping it 180\u25e6 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.
\n6. 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.
\n7. After grinding, quickly add the prepared buffer and start the protocol.<\/p>\n
PART IV: Protocol<\/strong><\/p>\n8. Put some 3%CTAB to warm at 65\u25e6F with loose cap
\n9. Add 1 ml of CTAB-free buffer (cold) + 6 \u03bcl \u03b2-mercaptoethanol (mix done beforehand in the fumehood with a bit extra)
\n10. 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
\n11. Spin at 10.000 g for 10 minutes in the centrifuge (longer may be required, depending on your tissue)
\n12. Discard the supernatant under the fumehood in a trash beaker
\n13. (if you use particularly nasty tissue, you can repeat this step)
\n14. Add 500 \u03bcl of pre-warmed 3% CTAB + 5 \u03bcl of \u03b2-mercaptoethanol in the fumehood
\n15. Incubate at 65\u00b0C for 60 minutes
\n16. Let the tubes cool down a little bit
\n17. Add 500 \u03bcl of chloroform-isoamylalcohol 24:1 in the fumehood. SUPER CORROSIVE!
\n18. Vortex
\n19. Spin at full speed for 15 minutes. Meanwhile, label new 1.5mL tubes
\n20. Move the aqueous phase to the new tubes (1.5mL) under the fumehood. This must be done exactly right \u2013 it\u2019s probably the most important step. The aqueous phase is the liquid above the solid phase in the centre of the tube. Don\u2019t take all the liquid, and don\u2019t disturb the part that\u2019s 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.
\n21. Add 250 \u03bcl of NaCl 5M, mix fast
\n22. Add 500 \u03bcl of cold isopropanol (2-propanol), mix (have a tube ready in the freezer)
\n23. Leave at least 20 minutes or overnight at -20\u00b0C. You can take a break here if you want!
\n24. Spin at max speed for 15 minutes
\n25. Wash pellet in fumehood with 1 ml of cold 80% ethanol (prepare the mix from 100% and PCR-clean water)
\n26. Spin at max speed for 5 minutes
\n27. Repeat wash (no need to be under the fumehood anymore)
\n28. Dry pellet (by putting the tubes in the heatblock at 25 to 37 degrees)<\/p>\n
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.<\/p>\n
29. Resuspend in 400 \u03bcl of high salt TE + 2 \u03bcl of RNAse A (sometimes more RNAse is needed)
\n30. Incubate at 37\u00b0C for one hour, mixing occasionally until the pellet dissolves. Sometimes this is a LOT of mixing.
\n31. Add 800 \u03bcl of cold 100% ethanol
\n32. Spin at max speed for 15 minutes
\n33. Wash pellet with 1 ml of cold 80% ethanol. If the pellet is invisible, it doesn\u2019t 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.
\n34. Spin at max speed for 5 minutes
\n35. Repeat wash<\/p>\n
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 \u2013 1250 ng when the optional steps (29-35) were included, and more if they weren\u2019t.
\n37. 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\u2019t tell you what might be in the sample along with the DNA.<\/p>\n
Hazards<\/strong><\/p>\nIt 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!).<\/p>\n
Advice<\/strong><\/p>\nThe biggest problems with this protocol were:<\/p>\n
\u2022 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\u2019t properly removed. Sometimes it removes well, other times not.
\n\u2022 RNA contamination. We sometimes had lots of RNA in the samples if we skipped steps 29-35. If this isn\u2019t a problem for you, omit them. If it is, include them \u2013 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\u2019t always easy.
\n\u2022 Low yields. Sometimes we just didn\u2019t get much DNA. The good news is, this often seemed to be protocol-dependent rather than sample-dependent. When a sample didn\u2019t work out, trying the same sample again often solved the problem.<\/p>\n
Some suggestions for troubleshooting:<\/p>\n
\u2022 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.
\n\u2022 Try starting with different amounts of tissue. Use more if you have no DNA. Use less if you have a contamination problem.
\n\u2022 Be careful washing the pellet: don\u2019t lose all or part of it! Maybe spinning at colder temperatures (4\u00b0C? 10\u00b0C?) would help.
\n\u2022 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.
\n\u2022 Try using your newest tissue, collected whenever cambium is active near you (probably spring).<\/p>\n
Final note:<\/p>\n
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!<\/p>\n","protected":false},"excerpt":{"rendered":"
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) \u2013 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 […]<\/p>\n","protected":false},"author":34681,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[369483],"tags":[747559,658831],"class_list":["post-1548","post","type-post","status-publish","format-standard","hentry","category-techniques","tag-cambium","tag-dna-extraction"],"_links":{"self":[{"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/posts\/1548","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/users\/34681"}],"replies":[{"embeddable":true,"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/comments?post=1548"}],"version-history":[{"count":9,"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/posts\/1548\/revisions"}],"predecessor-version":[{"id":1560,"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/posts\/1548\/revisions\/1560"}],"wp:attachment":[{"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/media?parent=1548"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/categories?post=1548"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/blogs.ubc.ca\/aitkenlab\/wp-json\/wp\/v2\/tags?post=1548"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
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