Mesoplasma florum:Genomic DNA

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Extraction of genomic DNA from Mesoplasma florum

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.
• chlorform / 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 a 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
• Setup fresh 2 ml tubes with 800 μl of phenol / chloroform / isoamyl alcohol 25:24:1, transfer the supernatent, and mix carefully.
• Spin down at 17000g for 2 minutes
• Setup fresh 2 ml tubes with 800 μl of chloroform / isoamyl alcohol 24:1, transfer the supernatent, and mix carefully.
• Spin down at 17000g for 2 minutes
• Setup fresh 1.5 ml tubes loaded with 1 μl Novagen Pellet Paint NF and transfer the supernatent 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 prior to adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in 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

For buffers, see Qiagen Buffers.

• 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