HELP ! bacterial genomic DNA kit

Susanne Rohrer srohrer at immv.unizh.ch
Wed Apr 19 07:03:57 EST 2000


Here's our genomic DNA protocol:

Chromosomal DNA (guaranteed only for S. aureus)

Material

LB, or Brain Heart Infusion for really good yields from staph

Lysis Mix:
0.1M Tris-HCl, pH 7.5
0.1M EDTA
0.15M NaCl

10mg/ml Lysozyme
10mg/ml Lysostaphin (for S. aureus - lytic enzymes for other cells may
be substituted)

5% SDS in 50% EtOH

Phenol/Chloroform/Isoamyl alcohol

TE

Rnase, Dnase free

EtOH 100%/70%

5ml Glass tubes (Loewenstein)
sterile Glass rods

Harvest 20ml o/n culture (5min 15000 rpm in SS34)
suspend in 3ml lysis mix containing 5ul lysostaphin and 25ul lysozyme
stock
incubate at 37°C for 30 min or longer until soln turns viscous and more
transparent
add 300ul SDS/EtOH, votex 10 sec, leave atRT for 5 min
extract with 1ml Phenol/Chloroform, vortex 10 sec
centrifuge 10min, 18000 rpm in SS34
transfer upper phase into loewenstein or other tube with TRUNCATED
pasteur pipette (it should be viscous) - take care not to take along any
phenol/protein
carefully overlay with 2 volumes of EtOH

spool out DNA on glass rod for about a minute
DNA should be clear and visible on rod like egg white for a good yield

transfer rod to 4ml 70% EtOH in glass tube
wash 2x in 70% EtOH
Tip off on kleenex
Dissolve in 1ml TE
add 1ul Rnase and incubate at 37°C for 30min-1hr
guess amount on gel

I use about 1ul of a 1:10 dilution for PCR


"Frank O. Fackelmayer" wrote:

> Susanne Rohrer wrote:
>
> > Have you tried it "by hand"?
> >
> > If not, I can send you a protocol.
> >
> > Susanne
>
> Hi Susanne,
> Could you post the protocol here, please.
>
> Thanks,
> Frank





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