3.2.1. DNA extraction using CTAB
This protocol is for the extraction of DNA from bee abdomens and/or the thorax, using a lysis buffer containing CTAB, a compound that is able to separate polysaccharides from other cell materials. The choice of tissues avoids eye contaminants such as pigments, which can inhibit PCR and other downstream applications. The method can be scaled down for the extraction of Varroa destructor mites (see the BEEBOOK paper on varroa (Dietemann et al., 2013) for details on sampling) or bee embryos and up for larger larvae and pupae (see section 1.2. for their collection). Volumes should be adjusted accordingly based on sample volume (i.e. initial grinding in 5X sample volume of buffer, ca.25->200 ml). The subsequent two extraction protocols are simpler, but the CTAB procedure is excellent for problematic samples and is flexible in terms of tissue disruption, separation, and rescue of nucleic acids.
- Extract only the abdomen and/or thorax if possible. If a whole animal is extracted, use a Qiagen or similar column following manufacturer’s protocol for final purification of extracted DNA in order to reduce pigments that can inhibit genetic assays.
- Put tissue from a single bee in a 1.5 ml microcentrifuge tube.
- Add 500µl of CTAB + 2µl 2-mercaptoethanol
100mM Tris-HCl, pH 8.0
2% w/v hexadecyl-trimethyl-ammonium bromide (CTAB)
This buffer both stabilizes nucleic acids and aids in the separation of organic molecules. See MSDS as CTAB is a potential acute hazard.
- Homogenize with pestle.
- Add 50µg proteinase K and 25µl of RNase
While this step is optional, proteinase K improves yields by disrupting cell and organelle boundaries and is critical for extraction of DNA from many microbes.
- Vortex briefly to mix.
- Incubate at 55-65°C from several hours to overnight. Invert occasionally during incubation (e.g., once every 30 minutes for the first two hours).
- Centrifuge for 1 min at maximum speed (~14,000 rpm).
Unwanted tissue debris will form a pellet at the bottom of the microcentrifuge tube.
- Transfer liquid to fresh tube, leaving tissue debris pellet behind.
- Add equal volume phenol:chloroform:isoamyl alcohol (25:24:1).
- Invert several times (10-20 times) to mix then put on ice for 2 min.
- Spin at full speed (~14,000 rpms) for 15 min at 4°C.
- Transfer upper phase to fresh tube.
- Add 500µl cold isopropanol + 50µl 3M NaOAc.
- Vortex to mix, then incubate at 4°C > 30 min.
Samples can be stored at ambient temperature at this point for several days if needed for transport or timing, otherwise 4°C is best.
- Spin at full speed (~14,000 rpms) for 30 min at 4°C.
- Carefully decant liquid from DNA pellet.
- Add 1 ml 4°C 75% EtOH. Tap vortex briefly to loosen pellet.
- Spin at full speed for 3 min at 4°C.
- Decant liquid from pellet.
- Air dry pellet about10 minutes to evaporate all
residual traces of alcohol.
Do not over dry pellet, as it will be hard to resuspend.
- Resuspend in 50-100µl nuclease-free water (overnight at 4°C).
- Check DNA quantity and integrity on an agarose gel.
- First, prepare TBE gel buffer (an aqueous solution with a final working concentration of 45 mM Tris-borate and 1 mM EDTA). This is often prepared first as a ‘5x’ concentration comprised of 4 g Tris base (FW = 121.14) and 27.5 g boric acid (FW = 61.83) dissolved into approximately 900 mL deionized water. Add 20 ml of 0.5 M EDTA (pH 8.0) to this solution and adjust the solution to a final volume of 1l. Confusingly, the ‘working solution’ of this buffer for most uses is as 0.5x = a 1/10 dilution of the stock buffer.
- For a 1.5% agarose gel on a large-format gel rig, add 3 g of sterile agarose to 200 ml TBE buffer in a 500 ml or larger Erlenmayer flask, microwave at high heat for ca. 45 s (without boiling). For smaller gel rigs the volume of the gel can be from 50 to 100 ml. Take flask out and swirl vigorously, then heat in the microwave again until at full boil for 45 seconds, monitoring to avoid spillover. The agarose must fully dissolve so the liquid is perfectly clear
- Let the solution cool while swirling every minute until the flask can be held for several seconds without unbearable heat
- While hot, pipette in 10 µl ethidium bromide solution (EtBr, 0.5 mg/ml, used with caution as EtBr is a carcinogen and mutagen) and swirl until mixed
- Pour into a horizontal gel rig and insert plastic combs holding ca. 10 µl of sample each
- Let the gel solidify fully; gels can be wrapped in plastic wrap for longterm storage (overnight in place or for days at -4oC).
- Mix 5 µl of the extraction solution with 2 µl of a 40% weight/volume sucrose load buffer (made as 4 g sucrose and 25 mg bromophenol blue in 10 ml distilled water)
- Submerge gel in a rig containing 0.5 x TBE, remove gel comb and load the 7µl of sample/dye mix in separate wells using DNA molecular weight standards (e.g., 500 bp molecular ruler, www.biorad.com)
- Draw the DNA across the gel toward the anode/positive charge at ca. 100 V depending on the gel rig size and specifications.
- Monitor via the blue bromophenol blue stain movement (which tracks a DNA size fragment of ca. 300 bp in a 1.5% gel), stopping the gel and visualizing the DNA using ultraviolet light when it has progressed enough.
- DNA can also be quantified via a spectrophotometer such as the Nanodrop (www.nanodrop.com), following manufacturer’s protocol: Briefly, after calibration 1 µl of nucleic acid solution is placed onto a cleaned pedestal, the lid is closed and a reading is taken prior to cleaning by wiping the pedestal in preparation for the next sample . The machine will estimate concentration using the equation dsDNA: A260 1.0 = 50 ng/µl.
- Store at -20°C or below.