From: Gary Kucera <kucera~This email address is being protected from spambots. You need JavaScript enabled to view it.>
Newsgroups: bionet.molbio.methds-reagntsSubject: Re: mouse tail DNA extraction kits
Date: 13 May 1996 11:19:45 GMT
Organization: Glaxo Wellcome
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We have tried several kits and homemade methods and the protocol below
has given us the most consistent results:
Genomic DNA Prep
CELL LYSIS
• Add 600 ul of Cell Lysis Solution and 3 ul of Proteinase K (20 mg/ml)
to 1-2cm mouse tail tissue.
• Incubate >3 hours at 55oC or until tail is dissolved
RNase TREATMENT
• Cool to 37oC and add 3 ul RNase A Solution
• Incubate 15-60 minutes at 37oC
PROTEIN PRECIPITATION
• Cool Sample to room temp and add 200 ul of Protein Precip. Solution
• Vortex thuroughly to mix
• Centrifuge for 3 minutes
DNA PRECIPITATION
• Transfere supernatant containing the DNA to a fresh eppie
• Add 600 ul 100% isopropanol and invert several times to mix until DNA
forms a visible clump
• Centrifuge for 4 minutes and CAREFULLY remove ethanol.
• Wash pellet with 70% ethanol
DNA HYDRATION
• Add 100 ul DNA Hydration Solution and allow DNA to resuspend either at
65o C for 1 hour or overnight at room temp
• Store at 4o C
Cell Lysis Solution:
10mM Tris, pH 8.2
1mM EDTA
0.67% SDS
Protein Precip Solution:
6.5 M NH4oAc
Hydration Solution
TE, pH 7.4-7.6
----------------------------------------
We have also used the following protocol (although not as extensively)
but the results do not seem to be as consistent (ie poor amplification
of DNA target using identical primers when compared to the prep outlined
above). But the protocol is fast:
Quick Tail DNA Prep for PCR
• Place 2 mm tail section into 200 ul 1x PCR buffer (Contains 0.45% v/v
NP-40, 0.45% v/v Tween-20)
• Add 3 ul ProK solution and incubate at 55 C until digestion is complete
(about 1-2 hrs)
• Heat samples in boiling waterbath to inactivate ProK (about 5 min.)
• Vortex well then Spin 2 minutes
• Use approx. 2 ul of this prep in a 50 ul PCR reaction
____________________________________
Gary T. Kucera, Scientist
Molecular Biology/Transgenics Group
GlaxoWellcome Research, Inc.
Five Moore Drive
Research Triangle Park, NC 27709
