Mesoplasma florum:Genomic DNA

Extraction of genomic DNA from Mesoplasma florum

This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.

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

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.