DNA extraction with cetyl-trimethylammoniumbromide (CTAB)

1. Subject

 For whole genome sequencing it is important to obtain high molecular weight DNA (HMW-DNA). There are several protocols published for extracting DNA from micro-organisms, including fungi and yeast. This protocol is a simple protocol in which DNA can be obtained within a day and suitable for Nanopore sequencing. It does not include enzymatic lysis or grinding with liquid nitrogen. DNA extracts are treated with RNAse to get RNA-free DNA samples. The procedure is based on a micro-prep method and does not require high loads of biomass. It seems, that best results are obtained with young cultures and when grown on suitable media. For all yeasts this is established by growing them on media (e.g. GYPA, glucose yeast peptone agar) with 2.9% NaCl.

2. Principle

CTAB extraction methods are a common way of extracting DNA from fungi, including yeasts. Mechanical lysis is used, but not severely. Most of the work is done by incubating tissue in a CTAB 2% buffer with the presence of proteinase K at an optimal temperature. CTAB is destroying the cross links in the fungal cell wall and dissolving cell and nuclear membranes. 

3. Reagents and materials

3.1       cetyl-trimethyl ammonium bromide, CTAB ([57-09-0], Sigma H6269)
3.2       Tris (Sigma 252859, [77-86-1])
3.3       NaCl (Sigma S9888,  [7647-14-5])
3.4       Na-EDTA (Sigma 1084211000, [6381-92-6])
3.5       proteinase K (Promega V3021)
3.6       glass beads 2 mm (VWR 1.04014.0500)
3.7       vials 2.0 ml (BIOzymTC B91201)
3.8       caps (BIOzymTC B91300)
3.9       isopropanol -20°C (Sigma I9516, [67-63-0])
3.10     ethanol 70% -20°C
3.11     vials 1.5 ml (Sarstedt )
3.12     ethanol 96% (Sigma 1.00971, [64-17-5])
3.13     HCl 37% (Sigma 1.01834, [7647-01-0])
3.14     RNAse cocktail mix (Invitrogen AM2286)
3.15     TE-buffer, low concentration (IDTE pH8.0, 11-05-01-09)
3.16     iso-amylalcohol (Sigma 8.22255, [30899-19-5])
3.17     chloroform (Sigma 288306, [67-66-3])

4. Solutions

4.1       CTAB 2% extraction buffer:
Add 2.42 g Tris [3.2], 8.2 g NaCl [3.3], 0.74 g Na-EDTA [3.4] and 2 g CTAB [3.1] to 80 ml ultrapure water. Adjust pH to 8 with 1 N HCl. Heat the solution (55-65°C)  to dissolve Na-EDTA and CTAB if necessary. Adjust volume to 100 ml with ultrapure water. Autoclave 15 min. at 121°C. Filter sterilization is also possible. Save at room temperature.
4.2       Proteinase K:
Dissolve 100 mg proteinase K [3.5] in 4 ml sterile demi-water. Make aliquots of 250 µl in 1.5 ml vials. Store at -20°C.
4.3       Acid ethanol:
Mix concentrated HCl [3.13] with 96% ethanol [3.12] in a 1:1 ratio [6.1].
4.4       SEVAG:
Mix chloroform [3.17] and iso-amylalcohol [3.16] in a 24:1 ratio, e.g. 240 ml chloroform and 10 ml iso-amylalcohol [6.5]
4.5       CTAB 5%:
Dissolve 2.5 g CTAB [3.1] in 50 ml demi-water by heating to 60-65°C. Solution can be filter-sterilized or autoclaved.

5. Protocol

The stepwise protocol can be downloaded as an Excel file:
Previous version

Prepare glass beads:
wash (incubate) glass beads in acid ethanol [4.3] for 1 day. Rinse the beads 3 times with sterile demi-water. Discard water and dry the beads at room temperature or in an incubator. Collect the beads in a glass bottle.
Prepare extraction vials:
add 12-20 glass beads in a vial [3.7] and autoclave at 121°C for 15 min.; add 490 µl CTAB buffer [4.1] and 10 µl proteinase K [4.2]. It is possible to make a mastermix of CTAB buffer and proteinase K in a 49:1 ratio and add 500 µl to a glass beads filled tube.

5.1 Switch on water bath (or heating block) and set to 65°C.
5.2 Add fungal material (4-16 cm²) or a 10 µl loop for yeasts [6.2] to an extraction vial.
5.3 Incubate for 30-60 min. at 65°C . Mix every 10-15 min.
5.4 Add 250 µl CTAB 5% buffer [4.5] and vortex 10 min. or Qiagen Tissuelyser: 3 min./20^-s (90 sec./30^-s).
5.5 Incubate for at least another 60 min. at 65°C. Mix every 10-15 min. [6.3].
5.6 Let it cool to room temperature on ice (considering the amount it will take a minute).
5.7 Add 750 µl SEVAG [4.4] and shake for 2 min. to rinse the lysate [6.4].
5.8 Centrifuge in a microcentrifuge at max. speed for 10 min.
5.9 Collect only the (upper) aqueous layer [6.6], ~650 µl in a clean, labeled vial [3.11].

      The remains are chemical waste and should be discarded in a proper way.

5.10 Add 0.55x the collected volume of ice cold iso-propanol [3.9] to [5.9]. E.g. if 650 µl is collected, then add 360 µl iso-propanol.
5.11 Mix by inverting the tubes about 20 times. DO NOT VORTEX [6.7].
5.12 Centrifuge in a microcentrifuge at 12,000 rpm for 10 min. to pellet DNA/RNA mixture.
5.13 Pour off the iso-propanol. Be sure not to loose the pellet.
5.14 Pipet 1 ml ice cold ethanol 70% [3.10] and mix by flipping the vials [6.8].
5.15 Pour off the ethanol. Be sure not to loose the pellet.
5.16 Dry the samples by air or use a SpeedVac concentrator [6.9].
5.17 Add 50 µl TE-buffer [3.15] containing 0.5 µl RNAse [3.14] to the pellet [6.11].
5.18 Incubate at 37°C for 45-60 min. with regular tapping to resuspend the pellet.
5.19 Store the extracts at -20°C [6.10].

6. Remarks

6.1 Use HCl 37% (very concentrated HCl ±12 N, fuming) in a fume hood. Add the concentrated HCl to ethanol, e.g. 250 ml ethanol 96% + 250 ml HCl.
6.2 The amount depends on the status of the mycelium (thin/thick). The loop should be richly filled with yeast cells. Fungal material should always be completely covered by extraction buffer.
6.3 This incubation step can be extended to 1.5-2 hrs. Some fungi even require a longer incubation time.
6.4 DO NOT VORTEX. Shake by hand or use a reciprocal shaker to establish a constant white emulsion. SEVAG should be handled in a fume hood. This step is critical and should be rather longer than shorter.
6.5 Use protective clothing and suitable gloves when handling chloroform. Always prepare this mixture in a fume hood.
6.6 After centrifugation 3 layers are visible, the lower layer containing chloroform, middle layer with denatured proteins and cell debris, and the upper aqeous layer (buffer) containing DNA and RNA. It goes without saying, that ONLY the upper layer should be transferred.
6.7 Vortexing or pipetting will destroy the integrity of the genome. The samples can be kept at -20°C (even over night) to increase yield, but it will also precipitate unwanted compounds.
6.8 If the pellet comes loose, centrifuge for about 2 min. to collect the pellet.
6.9 Do not overdo it. When too dry, it will damage the DNA or makes it hard to resuspend.
6.10 Several quality checks can be performed by 0.8% agarose gel electrophoresis, Nanodrop and/or Qubit measurement.
6.11 The amount of TE-buffer also depends on the size of the pellet. To obtain a high concentration add as little as possible, but enough to dissolve the pellet. It is always possible to add more.

7. Literature

7.1 Navarro-Muñoz, J.C., de Jong, A.W., Gerrits van den Ende, B. et al. The High-Quality Complete Genome Sequence of the Opportunistic Fungal Pathogen Candida vulturna CBS 14366T. Mycopathologia 184, 731–734 (2019).
https://doi.org/10.1007/s11046-019-00404-0

8. Graphs and tables

8.1 CTAB 2% buffer with prepared liquids