N.crassa Genomic DNA

Matthew Sachs msachs at ADMIN.OGI.EDU
Tue Apr 5 15:30:48 EST 1994


Neurospora crassa DNA mini prep
(salt-detergent and TCA/EtOH)

based on previous procedures; protocol written by Michael Freitag, in my
laboratory (freitag at admin.ogi.edu).


(1)     grow standing cultures in 25 or 50 ml of 1x Vogel's with 1.5%
sucrose in 125 or 250 ml Erlenmeyer flasks at RT for 2 or 4 days (yields
ca. 1.5 g wet mycelial weight; avoid aerial hyphae and onset of
conidiation)

(2)     harvest cells onto Whatman filter paper (# 1 or 541) in Buchner
funnel, determine wet weight, transfer into 15 ml Falcon (#2059) tubes (or
equivalent, spatula must fit in bottom), freeze -80o C for at least 20 min

(3)     lyophilize overnight, pulverize (vortex with spatula for ca. 30 sec)

(4)     suspend samples in salt-detergent solution by adding enough liquid
(usually 1 ml for 1 g  wet weight) to make a thick suspension after
vortexing at high speed for 20 sec

(5)     incubate at RT for 20 min while mixing end-over-end on rotator

(6)     spin in Sorvall SS-34 at 8000 rpm for 10 min, this usually yields
600 ul of spnt

(7)     collect clear, slightly orange spnt and transfer 300 ul per
Eppendorf tube

(8)     add 1.2 ml of TCA/EtOH and mix gently by inversion

(9)     precipitate at -20oC for at least 30 min (may be stored at this stage)

(10)    pellet nucleic acids by 15 sec microfuge spin, aspirate spnt, wash
pellet with 300 ul of 70% EtOH, spin for 15 sec, aspirate spnt, dry in
speed vac

(11)    resuspend nucleic acids in 100 ul of 10 mM NH4OAc, mix gently (it
takes some time for pellets to dissolve); add 100 ul of 0.3 mg/ml RNase A
in 10 mM NH4OAc, mix by pipetting up and down, incubate at 50o C for 1 h,
every 15 min vortex gently to resuspend pellet

(12)    add 200 ul of chloroform, vortex, spin in microfuge for 5 min,
transfer spnt to new tube (at this point spnts of two tubes may be
combined)

(13)    add 107 ul of 7.5 M NH4OAc, then 0.8 ml isopropanol, mix well by
inversion, spin immediately in microfuge for 25 sec (later precipitate is
mostly unwanted junk), aspirate spnt, wash pellet with 300 ul of 70% EtOH,
aspirate spnt, briefly dry in speed vac (if dried too hard DNA ressuspends
only poorly)

(15)    resuspend pellet in 100 ul TE buffer overnight at 4o C

(16)    yields 40 to 90 ug of genomic DNA per culture,
        digest to yield 1 to 2 ug of DNA per lane


SALT DETERGENT SOLUTION

-  dissolve in this order and completely:
                Na Deoxycholate                                            
      2.00 g
                Brij 58 (polyoxyethylene20cetyl ether)                     
      5.00 g
                NaCl                                                       
            58.44 g
                in 350 ml H2O
-  adjust to 500 ml, store at 4o C


TCA/ EtOH SOLUTION

-  make 4.5 M TCA by dissolving  41.7 g NaTCA salt (Aldrich #19,078-0) in
H2O until 50 ml are reached (density is 1.43 g/ml, i.e. wt of 50 ml should
be 71.5 g)
-  add 50 ml EtOH, store at 4o C (precipitate will appear upon mixing and
settles; don't worry, but don't mix it with the clear solution when using)


RNase SOLUTION

-  10 mg/ml RNase stock is diluted into 10 mM NH4OAc to yield 0.3 mg/ml
RNase A (30 ul RNase A for 1ml of 10 mM NH4OAc)


References:

1.      Oakley, C.E. et al. 1987. Cloning of the ribo B locus of
Aspergillus nidulans. Gene 53:293-298.

2.      Summerton et al. 1983. A rapid method for preparation of bacterial
plasmids. Anal. Biochem. 133:70-84

3.      pers. commun. E. Selker lab, University of Oregon

4.      pers. commun. Zongli Luo, Matthew S. Sachs and Duane T. Mooney, OGI





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