Materials
- ATCC 1161 culture medium
- TE
- 10% SDS solution
- Proteinase-K 20 mg/ml solution
- 5 M NaCl solution
- CTAB / NaCl solution
- CTAB 10%, NaCl 700 mM
- dissolve 4.1 g NaCl in 80 ml water, and slowly mix and add 10 g CTAB (hexadecyltrimethylammonium bromide) heating to 65° if necessary. Bring the solution to 100 ml.
- chloroform/isoamyl alcohol 24:1
- phenol/chloroform/isoamyl alcohol 25:24:1
- Novagen Pellet Paint NF
- isopropanol
- 70% ethanol
CTAB Protocol
- Culture 10 ml of medium 1161 infected with Mesoplasma florum in 15 ml centrifuge tubes at 30° without shaking.
- Transfer 2x 1.8 ml into two 2 ml tubes. Spin down at 17000g for 2 minutes and retain the pellet.
- Resuspend the pellet by vortexing first, then adding 567 μl of TE.
- Add 3 μl of proteinase-K 20 mg/ml and 30 μl of SDS 10% solution, mix, and incubate at 37° for 1 hour
- Add 100 μl of 5 M NaCl and mix (critical before adding CTAB)
- Add 80 μl of CTAB / NaCl solution, mix
- Incubate 10 minutes at 65°
- Add an equal volume (800 μl) of chloroform/isoamyl alcohol 24:1, vortex, and spin down at 17000g for 2 minutes
- Set up fresh 2 ml tubes with 800 μl of phenol/chloroform/isoamyl alcohol 25:24:1, transfer the supernatant, and mix carefully.
- Spin down at 17000g for 2 minutes
- Set up fresh 2 ml tubes with 800 μl of chloroform/isoamyl alcohol 24:1, transfer the supernatant, and mix carefully.
- Spin down at 17000g for 2 minutes
- Set up fresh 1.5 ml tubes loaded with 1 μl Novagen Pellet Paint NF and transfer the supernatant into those tubes.
- Add 0.6 volumes of isopropanol and chill in the -80 freezer for 30 minutes
- Spin down at 17000g for 20 minutes and remove the supernatent carefully retaining the blue pellet.
- Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.
- Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 μl tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 μl.
- Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.
- Redissolve the DNA pellet in 100 μl TE (this will take an hour or so).
- Spin down the tube briefly and measure OD and 260/280 ratios.
- Expect around 500 ng/μl concentration (total 50 μg).
A critical element is the NaCl concentration before adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in the solution.
Reference
- PMID 7433111
- This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.
Qiagen Genomic Tip protocol
- Grow 40 ml cultures of the Mesoplasma species at 30°C in 1161 medium.
- Pellet cells at 5000 x g for 10 minutes
- Resuspend the pellet in 11 ml of buffer B1 (with RNAse)
- Add 500 μl of proteinase-K solution, 20 mg/ml (10 mg)
- incubate at 37°C for at least 30 minutes
- Add 4 ml of buffer B2 and mix or vortex briefly
- Incubate at 50°C for 30 minutes; the lysate should become clear
- If necessary, pellet particulates
- Save a 300 μl sample 1
- Equilibrate a genomic tip 500/G with 10 ml of buffer QBT and allow flow through
- Vortex the sample for 10-20 seconds to reduce viscosity
- Sample should flow through, or can be diluted with buffer QBT before loading
- limit flow to 20-40 drops/min under pressure
- save a 1200 μl sample of the flow through — sample 2
- Wash the column with 15 ml of buffer QC twice or more
- save a 600 μl sample of the flow through — sample 3
- Elute with 15 ml of buffer QF prewarmed to 50°C
- save a 600 μl sample of the elution — sample 4
- precipitate DNA by adding 10.5 ml (0.7 volumes) of isopropanol
- invert the tube several times and recover by spooling on a glass rod or
- Centrifuge at 5000 x g for 15 minutes at 4°C, wash with 70% ethanol
- dissolve in 100-200 μl of TE pH 8.0 on a shaker at 55°C or overnight
- For diagnosis of problems:
- precipitate the DNA from the samples 1-4 taken above with 0.7 volumes of isopropanol
- wash with 70% ethanol
- Resuspend in 20 μl TE
- Run 10 μl on a 0.5% agarose gel