Mesoplasma florum:Genomic DNA

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 4: Line 4:
-
Materials:
+
==Materials==
* ATCC 1161 culture medium
* ATCC 1161 culture medium
Line 20: Line 20:
* 70% ethanol
* 70% ethanol
 +
==Protocol==
-
Culture 10 ml of medium 1161 infected with Mesoplasma florum in a 15 ml centrifuge tubes at 30° without shaking.
+
* 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).
-
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.
+
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 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)
+
==Reference==
 +
* PMID 7433111
 +
* This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.
-
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.
 
-
Literature reference: PMID 7433111
 
[[category:Mesoplasma florum]][[category:protocol]]
[[category:Mesoplasma florum]][[category:protocol]]

Revision as of 14:20, 9 September 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
  • 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.
Personal tools