Mesoplasma florum:Genomic DNA

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Extraction of genomic DNA from Mesoplasma florum
Extraction of genomic DNA from Mesoplasma florum
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Culture 10 ml of medium 1161 infected with Mesoplasma florum in two 15 ml centrifuge tubes at 30° without shaking.
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This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.
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Spin down at 3000g for 10 minutes and retain the pellet.
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Materials:
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Resuspend the pellet by vortexing first, then adding 1 ml of TE.
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* ATCC 1161 culture medium
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* TE
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* 20% SDS solution
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* Proteinase-K 20 mg/ml solution
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* chlorform / isoamyl alcohol 24:1
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* phenol / chloroform / isoamyl alcohol 25:24:1
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* isopropanol
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* 70% ethanol
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Add 3 μl of proteinase-K 20 mg/ml, mix
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Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.
-
Add 15 μl of SDS 20% solution
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Transfer 2x 1.8 ml into two 2 ml eppendorfs.  Spin down at 8000g for 2 minutes and retain the pellet.
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Incubate at 37° for 1 hour
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Resuspend the pellet by vortexing first, then adding 567 μl of TE.
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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 100 μl of 5 M  NaCl and mix (critical before adding CTAB)
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Incubate 10 minutes at 65°
Incubate 10 minutes at 65°
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Add an equal volume (1.2 ml) of chloroform / isoamyl alcohol 24:1, vortex and spin down at 7000g for 6 minutes
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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.
Remove the supernatent to a fresh tube and add an equal volume of phenol / chloroform / isoamyl alcohol 25:24:1 and mix carefully.
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Redissolve the DNA in 100 μl TE (this will take an hour or so).  Roll the liquid in tube to wet the entire surface.
Redissolve the DNA in 100 μl TE (this will take an hour or so).  Roll the liquid in tube to wet the entire surface.
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Spin down the tube briefly and transfer the DNA to a small tube.
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Spin down the tube briefly and measure OD and 260/280 ratios.
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Measure OD and 260/280 ratios.
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Revision as of 10:18, 31 August 2007

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
  • chlorform / isoamyl alcohol 24:1
  • phenol / chloroform / isoamyl alcohol 25:24:1
  • 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 7000g for 6 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 7000g for 6 minutes

Remove the supernatent to a fresh tube and add 0.6 volumes of isopropanol; DNA should precipitate as a clear/white pearly substance in solution. Hook the DNA with a sterile pasteur pipet, drain, and tranfer to a fresh tube, or alternatively, spin down at high speed and pour off the supernatent.

Fill the tube with 70% ethanol to wash, and spin down at 7000g for 6 minutes.

Remove the ethanol carefully leaving the white pellet. Let the tube air dry for 1/2 hour until the ethanol odor is no longer present

Redissolve the DNA in 100 μl TE (this will take an hour or so). Roll the liquid in tube to wet the entire surface.

Spin down the tube briefly and measure OD and 260/280 ratios.

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