Quick DNA Prep for PCR?
jakku at mrna.tn.nic.in
Thu Aug 23 13:36:28 EST 2001
I used to use this protocol for extraction of very good quality DNA from
leafhoppers during my MSc. days several years back. It shouls work out for
beetles too. I am not sure of the reference, but I can dig it out for you if
you really need it.
1. The leafhoppers crushed in a glass tissue-homogeniser in ice cold LEB
(20mM Tris-HCL (Ph8.0), 4 mM EDTA, 0.5M NaCl, 0.2% 2-Mercaptoethanol).
2. SDS added to a final conc. of 1%
3. Incubated at 65C for 5 min. and potassium acetate added to 1.25 M final
4. tubes were incubated on ice for 30 minutes and centrifuged at 15000g for
5. Supernatants extracted once with an equal volume of phenol:chloroform
and twice with chloroform (I don;t think addition of Isoamylacohol would
cause any problem).
6. Nucleic acids were ethanol precipitated (2.5Volumes). and collected by
centrifugation at 12,000g for 20min.
7. Given a 70% wash and airdried or vacuum dried for a few min. and then
dissolved in appropriate volumes of TE (10mM Tris (ph8.0), 1mM Edta).
----- Original Message -----
From: "J. Rinehart" <jrineharNOSPAM at snake.eou.edu>
To: <methods at hgmp.mrc.ac.uk>
Sent: Thursday, August 23, 2001 11:07 PM
Subject: Quick DNA Prep for PCR?
> Hello all:
> My colleagues and I have identified a PCR marker distinguishing two
> species of closely-related bark beetle: Dendroctonus pseudotsugae
> (Douglas Fir beetle) and D. rufipennis (spruce beetle). When we trap for
> these beetles, often we get individuals in the traps that have been
> damaged (usually by predators that follow the pheromone into the trap
> along with their prey) to the point of being unable to identify them
> morphologically, hence the usefulness of this marker. However, the
> unidentifiable individuals often number in the hundreds, and so we are
> looking for a rapid, non-labor intensive method of getting DNA out of
> these to run our PCR reaction on.
> We tried a simple crude homogenate that I never expected to work, and it
> did not disappoint. We tried boiling the ground-up beetles to break up
> lipid bilayers and release the genomic DNA; this also did not work.
> Standard extraction methods (using Promega's Wizard genomic prep kit)
> work very well, but this is way too labor and time intensive.
> Sonication is not a likely option for us. Could we add a small amount of
> detergent, such as Triton-X, to the crude homogenate or to the PCR
> reaction? Any suggestions are welcome. Thanks.
> John Rinehart
> Eastern Oregon University
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