Mycobacterial DNA isolation

Laura Via LEVIA at VTVM1.CC.VT.EDU
Wed May 11 18:17:16 EST 1994


Sorry to post this to everyone but the address given would not work
from this address. Warning, I cannot control the XEDIT editor,
lines may capitalize themselves for no known reason.
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Date:         Wed, 11 May 94 16:16:36 EDT
From:         "Laura E. Via <LEVIA at VTVM1>
Subject:      Re: Purification of Mycobacterium DNA
To:           Jerry Grandoni <grandoni at soma.UMDNJ.EDU>
In-Reply-To:  Your message of 10 May 94 17:28:50 GMT


Hi!  Don't you love the myco's cell surface!

THERE ARE LOTS OF MYCO DNA PREPS, I EVEN HAVE A FEW METHODS IN REVIEW.

MY SUGGESTION IS TO LOOKUP METH. ENZ. VOL FROM 2 YEARS AGO. BLOOM AND
JACOBS HAD A NICE METHOD USING THE MINI-BEAD BEATER FROM BIOSPEC PRODUCTS
WELL AS METHODS FOR ELECTROPORATION AND PLASMID PREPS. I'M DOING A POSTDOC
in a virology lab right now so my files are at home. If you can't find the
ISSUE, EMAIL ME.

here is my favorite "quick" method.

-grow cells as usual, 48h before harvest add additional 5-10% enrichment to
increase growth rate again.
-SPIN DOWN, RESUSPEND IN 50MM TRIS TCL PH 8, 5 MM EDTA, 100 MM NACL. ADD
1 gram acid washed 1mm glass beads, and beat in above machine for 1 min.
we use 2 ml tubes from starset.
-add rnase, incubate for 20 min at 43 c, add proteinase k for same at 50 c.
-transfer beads and all into larger tube and phenol extract, follow with
phenol chloroform extractions until no white ppt after spin. chloroform
extact. add nh4oac to 2.5m final conc. mix, sit on ice 30 min.
transfer super. to new tube even if no pellet is visible (this helps
get digestable dna).
-ADD 2-2.5 VOL 95% ETOH, INVERT GENTLELY TO MIX PHASES AND USE PASTEUR
PIPET HOOK TO FISH OUT DNA CLOT (OR SPOOL), RINSE IN 70% ETOH.
-RESUSPEND IN TE, HEAT 37 C TO DISSOLVE.
FOR LIBRARIES I'D USE THE J.J MCFADDEN METHOD (MOL. MICRO VOL 2 ?)

I HAVE ALL KINDS OF REFS AND HINTS-IF YOU NEED THEM, I'LL LOOK THEM UP.

LAURA E. VIA  <LEVIA at VTVM1.CC.VT.EDU>
RESEARCH ASSOC.
VA TECH (HOME OF J.O. FALKINHAM AND HIS AVIUM-LOVING CREW)
phone 703 231-7084
F.O. FALKINHAM <JOFTOPSM at VTVM1.CC.VT.EDU>
from my address. Also please note, I am not an expert with xedit.



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