Genomic DNA Isolation Procedure for Nostoc punctiforme                                                5/2000


This procedure should yield high molecular weight (greater than 50 kb) genomic DNA that is clean enough 
for standard molecular manipulations such as enzyme digestions.

Reagents required:
5M NaCl
TE (10 mM Tris + 1mM EDTA, pH=8)
Lysozyme (20 mg/ml in TE, make up fresh)
0.5M EDTA, pH=8
20% SDS
Proteinase K (2 mg/ml in TE, make up fresh) 
CTAB solution (10% CTAB + 0.7 M NaCl)
Phenol:chloroform (1:1, v/v)
Chloroform
3M sodium acetate (pH=5.2)
95% ethanol
70% ethanol

Procedure:

1.  Pellet the culture by centrifuging at 1,000 x g (maximum rpm in clinacial centrifuge) for 5 min in a 15
or 50 ml plastic centrifuge tube.  As much as 600 ug of Chl a (~ 6 x 108 cells) may be processed at one time.

2.  Wash the pellet twice with 5M NaCl for removal of polysaccharides.

3.  Resuspend by vortexing the pellet in 1.0 ml total volume with TE.

4.  Add 1.0 ml of lysozyme solution.  Invert gently, then incubate at 37C for one hour.  Invert periodically during 
incubation.

5.  Add 0.5 ml of 0.5 M EDTA.

6.  Add 1.0 ml of proteinase K solution and 100 ul of 20 % SDS.  Invert gently to mix.  Incubate at 37C for 
an hour, inverting gently several times during incubation.

7.  Add 1/6 x vol (600 ul) of 5M NaCl, mix.

8.  Add 1/8 x vol (450 ul) of CTAB solution, mix.  Heat to 65C for 10 min.

9.  Pellet the cell debris by centrifugation at 13, 000 x g for 5 min at room temperature.  Decant the supernatant 
gently into a new tube.  Be careful to avoid the disk of cell debris that sometimes forms on top of the liquid.  
Be sure the centrifuge is at room temperature and not cooled, as DNA will pellet at cooler temperatures as well.

10.  Extract once with chloroform.  Transfer aqueous phase to a new tube.

11.  Precipitate the DNA with 2 x vol of 95% ethanol.  Stringy DNA molecules should be macroscopically visible 
at this point.  Pellet at 13,000 x g for 5 min.  

12.  Dissolve DNA pellet with 500 ml of TE and transfer to a microfuge tube.  Extract with an equal volume 
of phenol:chloroform.  Repeat extractions until the interface is clear.  For highest yields of DNA back
extract all organic phases.

13.  Precipitate DNA by adding 1/10 x vol of 3 M sodium acetate and 2 x vol of 95% ethanol.  Wash the pellet
with 70% ethanol.

14.  Dissolve final DNA pellet in desired volume of TE and digest with RNase if desired.