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Autoclaves/DNA revisited

Sat Aug 24 15:43:07 EST 1996

Hello again,

    On 5 August, I posed the question of whether autoclaving could destroy
DNA; thanks to all who replied.  To conclude (or perhaps further) this
discussion, I have written the follow-up comments/synopsis below:

      By "destroy", I meant that the DNA would no longer be amplifiable in a
PCR reaction.  A few people apparently thought I was asking whether the dsDNA
would be "denatured" by an autoclave.  From a standpoint of a PCR reaction, I
don't think the issue of whether autoclaving will melt the DNA strand is even
pertinent; after all, denaturation is one of the 3 cycles in a PCR reaction.
Destruction of the DNA (in this context) would need to either fragment the DNA
into extremely small pieces or change the DNA is some way that makes it
impossible to copy.

    I have compiled most of the responses below.  The general consensus (at
least for liquids) is that autoclaving cannot guarrantee that contaminating
DNA will be destroyed -- no matter how long the autoclave cycle is.
Apparently, the DNA will be sheared, but amplifiable fragments can still
remain.  Some suggested that the fragments would not be >250 bp while others
indicated that much larger fragments could remain.  One group in Texas
chose to cross-link the DNA by a 5 min. exposure of 0.5 ml tubes (100 ul vol)
to UV-light.

    Another approach was to use HPLC (0.2 um filtered) water, aliquoted into
small tubes.  Such water is "manufactured" under extremely clean conditions
and should be free of contaminating DNA.  Aliquoting w/o sticking anything
into the HPLC bottle is critical to keeping it free of DNA.  That approach
handles the water, but leaves the question of dissecting equipment and DNA
extraction equipment unanswered.  One person suggested treating such items
with bleach, stating that bleach definitely destroys DNA.

    Considering how difficult it is to resuspend DNA that has been overdried,
I suspect that autoclaving forceps, pestles, etc. can be effective as long as
the instruments are kept dry and autoclaved for at least 20 minutes.  In this
case, the DNA is probably not actually "destroyed", but rather becomes almost
impossible to resuspend -- which would effectively amount to being destroyed.

     If anyone has further thoughts on this topic, I'd appreciate hearing about
them (sherke at lsuvm.sncc.lsu.edu).  See the statements below for other peoples'
experiences with this issue.

Personal reply (w/holding name/email as I did not request permission to repost)
    I did not agree with some of the replies, especially those saying that
autoclaving would take care of your problem.  Sure, it will kill bacteria.
But, it won't do much to hurt the DNA.  We routinely use a sonicator to whack
DNA -- much worse than autoclaving -- but rarely does it fragment much below
500 bp or so.  DNA is tough.  Remember, even filtering won't get rid of
contaminating DNA.

Personal reply (w/holding name/email as I did not request permission to repost)
    I don't have specific information re autoclaving DNA and its effectiveness.
Sealed tubes may reduce the effectiveness of the autoclave; however, with
water in the tubes and the temperature reached by the autoclave (steam under
pressure), it should be effective.  Regarding autoclaving razor blades wrapped
in foil, if sealed to keep dry, any air pockets around the razor blade can
reduce the effectiveness of the process (it acts as an insulator), especially
if the blades are kept dry (heat with moisture is more effective than dry heat)

Replies posted on the net:

Ralph Bernstein (ralph at ccit.arizona.edu):
    I have heard that genomic dna is broken up, but that small pieces (i.e.,
perfect PCR contaminant sized) is still ok.

Miller at Csmc.edu:
    I bet that pH of the DNA solution is important, as is the purine content.

Michael Freitag (freitag at morel.uoregon.edu)
    I had heard the same things as you with regard to autoclaving pipet tips,
etc.  When I tested this, I found that autoclaving does not do the job complete
ly.  If you are really frantic about the possibility of contamination, use
bleach -- hypochlorite will definitely destroy DNA and seems to me the best way
to deal with contamination problems.
    As regards your side point, that one is true, as far as I know from my earl
y microbiology lab courses.  It depends on the mass of the object and the
thickness of the surrounding wall.  In your example, the tubes and the water ar
e most likely autoclaved (I've been doing this for years to prepare sterile wat
er to inoculate slants with conidia from Neurospora and other fungi and never
had trouble).  If, however, you take something like a wood block, say 4 by 4
inches, the center of the block will definitely not be sterile.

bgycsw at leeds.ac.uk (C.S. Wilding)
    I remember reading something on the bionet.molbio.rapds group a few years
ago in which someone had traced RAPD-PCR contamination back to the autoclave.
When their tubes/tips were autoclaved in the machine used for sterilizing
microbiological waste, then contamination occurred -- in another machine which
didn't have this function, the problem didn't occur.  Presumably, the a/c was
killing the bugs, but their DNA was being coated all over the inside of the a/c
and not being destroyed. Craig.

Michael Nedbal (nedbal at fmppr.fmnh.org)
    When I was working at Texas A&M, our entire building had problems of DNA
contamination in the water.  No amount of autoclaving destroyed the DNA --
confirmed by several labs.  Inevitably we ended up cross-linking the DNA with
exposure to UV-light.  A 5-min exposure to a closed 0.5 ml eppendorf tube (100
ul vol) did the trick.  I only had to do this when amplifying small <250 bp
fragments as the autoclave will shear larger fragments of DNA.  One more note
is that UV-exposure to mineral oil will inhibit PCR reactions (there was a
study in Biotechniques).

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