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ctab-dna-extraction.md

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Title: CTAB DNA extraction from problematic plant materials
Authors: Brant C. Faircloth
Address1: Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095, USA
Contact: Brant C. Faircloth
ContactEmail: brant.faircloth at gmail dot com
Copyright: 2010 Brant Faircloth
This work is licensed under a Creative Commons Attribution License.
http://creativecommons.org/licenses/by/3.0/us/
Keywords: CTAB, plants, DNA extraction, DNA purification

Summary

The following protocol is for CTAB extraction for plant materials + a pre-wash step for problematic templates (e.g. leaves). The pre-wash treatment derives from Li et al. 2007. The CTAB extraction is largely taken from the Soltis Lab DNA extraction protocol (which, itself, draws from Doyle and Doyle 1987 and Cullings 1992).

The pre-wash procedure helps deal with secondary plant compounds commonly found in plant leaves and other “non-soft” plant tissues. These include polysaccharides, polyphenolics, and tannins. 2-mercaptoethanol, PVP, and CTAB help remove these compounds from solution such that they are not carried over into the extracted DNA used in PCR (which they then inhibit, making you sad).

PVP absorbs polyphenols in solution (the PVP then being removed by pipetting) while 2-mercaptoethanol (AKA β-mercaptoethanol) helps to break disulfide bonds (i,e. proteins). CTAB, which is the other primary “ingredient” in the following protocol, binds polysaccharides, which are then removed from solution.

Goal

To remove PCR inhibitors from homogenates of plants prior to CTAB extraction

Main reference(s)

  • Cullings KC (1992) Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Mol Ecol 1: 233-240.
  • Doyle JD, Doyle JD (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin : 11-15.
  • Li JT, Yang J, Chen DC, Zhang XL, Tang ZS (2007) An optimized mini-preparation method to obtain high-quality genomic DNA from mature leaves of sunflower. Genet Mol Res 6: 1064-1071.
  • Soltis Lab DNA Extraction Protocol (accessed 07 July 2010)

Inputs

  • Homogenized plant tissue from above [pre-wash optional]

Outputs

  • Clean, high molecular-weight plant DNA for downstream analyses

Materials needed

  1. 1 M Tris-HCl
  2. 0.5 M EDTA
  3. CTAB | Amresco 0833-1KG

Detailed steps

  1. Mix CTAB buffer, PVP, and B-mercaptoethanol as follows

    Item 1X 30X 50X 100X
    CTAB Buffer 500.00 uL 15.0 mL 25.0 mL 50.0 mL
    PVP 0.02 g 0.6 g 1.0 g 2.0 g
    B-mercaptoethanol 2.5 uL 75.0 uL 125.0 uL 250.0 uL

  2. Add 500 uL of warm (55 C) CTAB buffer to a small amount (50-100 mg) of plant tissue in a mortar, and further grind the tissue for 2-3 minutes until it is a fine paste. Add approximately 50 mg of the paste to a microtube (and do the same with the remaining paste if you have some left) [in hood]

  3. Add 500 uL of CTAB buffer mixture from above to the plant homogenates

  4. Incubate samples at 55 C for 1-2 h. These can run overnight.

  5. [optional] Add 1.5 uL RNase A and incubate at 37 C for 15m

  6. Add 500 uL 24:1 chloroform:isoamyl alcohol to the microtube [in hood]

  7. Mix well by shaking

  8. Centrifuge tubes for 5-10 m at >10,000 RPM (or max for plate-centrifuge)

  9. Remove the aqueous phase by pipetting and transfer to a clean 1.5 mL microtube (do not pick up any of the interphase during pipetting). We typically only do this 1X as 2X doesn't return significantly better results, and pipetting around the interphase with 2X CI treatments is a little tough.

  10. Add 0.08 volumes of ice-cold 7.5 M ammonium acetate [in flammables fridge]. The total volume from the CI treatment should be ~ 400 uL, so you should be adding ~ 32 uL.

  11. Add 0.54 volumes of ice-cold 100% isopropanol (2-propanol) [in flammables freezer]. You should be adding around 234 uL.

  12. Mix well by shaking

  13. Place in freezer for 15 m to overnight. We have better yields overnight. But, there are more contaminants in overnight incubations.

  14. Centrifuge for 3 m at >10,000 RPM (or max for plate-centrifuge)

  15. Pour off supernatant gently, being careful not to dislodge the DNA pellet

  16. Add 700 uL ice-cold 70% ethanol and invert several times

  17. Centrifuge for 1 m at >10,000 RPM (or max for plate-centrifuge)

  18. Pour off supernatant, being careful not to dislodge the DNA pellet

  19. Add 700 uL ice-cold 95% ethanol and invert several times

  20. Centrifuge for 1 m at >10,000 RPM (or max for plate-centrifuge)

  21. Pour off supernatant, being careful not to dislodge the DNA pellet

  22. In the hood, invert the tube (gently!) on a kimwipe and let the pellet dry until the sample no longer smells of alcohol.

  23. Rehydrate DNA pellet with 50 uL TLE (Tris-'low' EDTA)

  24. [optional] Quantify samples using Nanodrop