FW: Freeze/Boil Cycling for Cell Lysis for rep-PCR

Stewart, Robert rob.stewart at labatt.com
Mon Feb 19 13:20:08 EST 1996


Greetings,

We have used thermocycling to lyse gram-positive bacteria and we have 
achieved high sensitivity PCR using this method.  Normally we place a single 
colony in 15-20 microliters of sterile water in a 0.5 ml microcentrifuge. 
 The suspension is then overlaid with sterile mineral oil and the tube 
placed into the thermocycler.  The thermocycler would then be cycled from 95 
degree Celsius to 8 degree Celsius 4 or 5 times.  Each temperature is held 
for 3 minutes. The final 'hold" temperature is 4 degrees.  After cycling the 
tubes are centrifuged in a microfuge at 10000 rpm for 2-3 minute.  10 
microlitres of the subsequent supernatant are then used for a 50 microliter 
(final) volume PCR.  We could detect less than 10 CFU bacteria using this 
method.  Interestingly, the sensitivity for yeast was worse, where the 
minimum number of cells we could detect was between 500 -1000 cfu yeast 
cells using this method.  I hope this is useful.
Cheers
RS


----------
From: 	Lemuel S. del Rosario[SMTP:ldelrosario at laba.tdh.texas.gov]
Sent: 	Friday, February 16, 1996 1:22PM
To: 	methods at net.bio.net
Subject: 	Freeze/Boil Cycling for Cell Lysis for rep-PCR

I am attempting to perform rep-PCR on gram-negative enteric bacteria for 
molecular subtyping purposes (Versalovic, et.al).The method I am using to 
liberate the DNA from whole cells is to do freeze/thaw cycles. Can anyone 
tell me specifics on how to do this crude DNA extraction, such as how 
many cycles are usual needed and how long to boil/freeze the samples.

Thanks,

Lemuel

Texas Department of Health
Bureau of Laboratories
Microbiological Investigation Section
ldelrosario at laba.tdh.state.tx.us






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