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
• 20% 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

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 eppendorfs. Spin down at 8000g 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 15 μl of SDS 20% 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 7000g for 6 minutes

Remove the supernatent to a fresh tube and add an equal volume of phenol / chloroform / isoamyl alcohol 25:24:1 and mix carefully.

Spin down at 17000g for 2 minutes

Remove the supernatent to a fresh tube and add an equal volume of chloroform / isoamyl alcohol 24:1 and mix carefully.

Spin down at 17000g for 2 minutes

Remove the supernatent to a fresh tube and add 1 μl of Novagen Pellet Paint NF, followed by 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution. Spin down at high speed and pour off the supernatent carefully retaining the blue pellet.

Fill the tube with 70% ethanol to wash, and spin down at 17000g for 6 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.

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.

Literature reference: PMID 7433111