Difference between revisions of "Mesoplasma florum:Genomic DNA"

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Extraction of genomic DNA from Mesoplasma florum
 
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.
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==Materials==
 
 
 
 
Materials:
 
  
 
* ATCC 1161 culture medium
 
* ATCC 1161 culture medium
 
* TE
 
* TE
* 20% SDS solution
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* 10% SDS solution
 
* Proteinase-K 20 mg/ml solution
 
* Proteinase-K 20 mg/ml solution
 
* 5 M NaCl solution
 
* 5 M NaCl solution
 
* CTAB / NaCl solution
 
* CTAB / NaCl solution
** CTAB 10%
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** CTAB 10%, NaCl 700 mM
** NaCl 700 mM
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** 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.
** dissolve 4.1 g NaCl in water, and slowly mix and add 10 g CTAB (hexadecyltrimethylammonium bromide) heating to 65° if necessary.
 
 
* chlorform / isoamyl alcohol 24:1
 
* chlorform / isoamyl alcohol 24:1
 
* phenol / chloroform / isoamyl alcohol 25:24:1
 
* phenol / chloroform / isoamyl alcohol 25:24:1
 +
* Novagen Pellet Paint NF
 
* isopropanol
 
* isopropanol
 
* 70% ethanol
 
* 70% ethanol
  
 +
==CTAB Protocol==
  
Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.
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* Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.
 
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* Transfer 2x 1.8 ml into two 2 ml tubes.  Spin down at 17000g for 2 minutes and retain the pellet.
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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* 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 30 μl of SDS 10% solution, mix and incubate at 37° for 1 hour
Resuspend the pellet by vortexing first, then adding 567 μl of TE.
+
* 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).
  
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)
+
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.
  
Add 80 &mu;l of CTAB/NaCl solution, mix
 
  
Incubate 10 minutes at 65&deg;
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==Reference==
 +
* PMID 7433111
 +
* This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.
  
Add an equal volume (800 &mu;l) 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.
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==Qiagen Genomic Tip protocol==
  
Spin down at 8000g for 2 minutes
+
For buffers, see [[Qiagen Buffers]].
  
Remove the supernatent to a fresh tube and add an equal volume of chloroform / isoamyl alcohol 24:1 and mix carefully.
+
* 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
  
Spin down at 8000g for 2 minutes
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* 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
  
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.
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* Wash the column with 15 ml of buffer QC twice or more
 +
* save a 600 &mu;l sample of the flow through -- sample 3
  
Fill the tube with 70% ethanol to wash, and spin down at 7000g for 6 minutes.
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* Elute with 15 ml of buffer QF prewarmed to 50&deg;C
 +
* save a 600 &mu;l sample of the elution -- sample 4
  
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
+
* 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
  
Redissolve the DNA in 100 &mu;l 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.
+
* 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
  
  
Literature reference: PMID 7433111
+
[[category:Mesoplasma florum]][[category:protocol]]

Latest revision as of 09:13, 11 November 2009

back to protocols

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