Quick DNA Prep for PCR?
dbell at qnis.net
Thu Aug 23 18:03:13 EST 2001
I am currently on a project isolating DNA from the head and thorax regions
of individual insects.
1) I am using 300ul of a basic CTAB extraction buffer, 50mM TRIS; 0.7M
NaCl; 10mM EDTA; 1% CTAB; 50Ul/ml 2-mercaptoethanol. Grind in utube with
sea sand using a dremmel
2) incubate at 65*C 1 hr,
3) equal volume chloroform:isoamyl alcohol extraction,
4) remove 200ul upper aqueous phase to new tube
5) equal volume isopropanol + 10% Na Acetate ppt
6) incubate at -20*C ~ 20 min
7) Centifuge and decant supernatent. Air dry pellet
8) dissolve pellet in 50ul TE,
10) add 2.5 volume ETOH and 1/10 volume Na Acetate
11) Repeat steps 6 - 8, ba-da-bing!
Those quicky preps can really degrade genomic DNA. If your marker is
small, sometimes they work, but if you have a marker >500bp you are better
off getting good quality, high molecular weight DNA to start with - IMO.
Hope this helps
USDA Agricultural Research Service
2021 South Peach Avenue
Fresno, CA 93727
(559) 453-3088 fax
On Thursday, August 23, 2001 10:37 AM, J. Rinehart
[SMTP:jrineharNOSPAM at snake.eou.edu] wrote:
: 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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