Mesoplasma florum:Genomic DNA

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&deg; 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&deg; 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 &mu;l of TE.
 * Add 3 &mu;l of proteinase-K 20 mg/ml and 30 &mu;l of SDS 10% solution, mix and incubate at 37&deg; for 1 hour
 * Add 100 &mu;l of 5 M NaCl and mix (critical before adding CTAB)
 * Add 80 &mu;l of CTAB / NaCl solution, mix
 * Incubate 10 minutes at 65&deg;
 * Add an equal volume (800 &mu;l) of chloroform / isoamyl alcohol 24:1, vortex and spin down at 17000g for 2 minutes
 * Setup fresh 2 ml tubes with 800 &mu;l 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 &mu;l 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 &mu;l 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 &mu;l tip to remove the remainder.  Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 &mu;l.
 * Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.
 * Redissolve the DNA pellet in 100 &mu;l TE (this will take an hour or so).
 * Spin down the tube briefly and measure OD and 260/280 ratios.
 * Expect around 500 ng/&mu;l concentration (total 50 &mu;g).

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&deg;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 &mu;l of proteinase-K solution, 20 mg/ml (10 mg)
 * incubate at 37&deg;C for at least 30 minutes
 * Add 4 ml of buffer B2 and mix or vortex briefly
 * Incubate at 50&deg;C for 30 minutes; the lysate should become clear
 * If necessary, pellet particulates
 * Save a 300 &mu;l 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 &mu;l sample of the flow through -- sample 2


 * Wash the column with 15 ml of buffer QC twice or more
 * save a 600 &mu;l sample of the flow through -- sample 3


 * Elute with 15 ml of buffer QF prewarmed to 50&deg;C
 * save a 600 &mu;l 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&deg;C, wash with 70% ethanol
 * dissolve in 100-200 &mu;l of TE pH 8.0 on a shaker at 55&deg;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 &mu;l TE
 * Run 10 &mu;l on a 0.5% agarose gel