Lysis Buffer Genomic DNA Buffer by lqz95924

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   C McKinney, Director                                    SOP Protocol
    PSU Transgenic Mouse Facility

                            Isolating DNA from Mouse Tail Clips

    Ref: PW, Laird et.al. “Simplified Mammalian DNA Isolation Procedure”,
        NAR 19: 4293, 1991.
    Note: Tail clips will be approximately 100 mm long.

     Procedure:
    1) Label microfuge tubes with mouse ear tag number. Put tail clips in 0.7 ml of Lysis
        Buffer (recipe below)+ proteinase K (dilute solution to 1mg/ml in Lysis buffer). Make sure
        tops are tightly closed. Rotate or shake overnight at 50 C.

    2) Next morning, label one set of microfuge tubes with mouse ear tag numbers.

    3) Vortex the tail digests. Spin samples in microfuge for 10 minutes at maximum speed.

    4) Hair and undigested debris may be in the precipitate. The supernatant may be colored but
       this does not effect the DNA isolation. Carefully transfer the supernatant (maybe thick and
       hard to pipette---use wide bore tips) to a clean tube with identical number. Add an equal
       volume (0.7 ml) of isopropanol (2-propane) to supernatant.

    5) Mix by inversion several times until white strands of DNA become visible.

    6) Spin the tubes at 10,000 x g for 5 minutes. Remove supernatant being careful not to disturb
       the DNA pellet in the bottom of the tube.

    7) Wash each DNA pellet with 0.5 ml of ice-cold 70% ETOH. Centrifuge and carefully remove all
       the ETOH wash (use a yellow pipette tip if necessary). Turn tubes upside down on paper
       towel to air –dry the DNA (do not over-dry or they DNA may be difficult to resuspend)

    8) When DNA pellets are dry (i.e., all traces of ETOH are gone), resuspend the DNA in 0.5 ml
       genomic DNA buffer (recipe below). Cap securely and rotate or shake DNA overnight at 37 C
       overnight to ensure complete DNA solution.

    9)  Next morning, read A260, A260/280 absorbance (usually takes a 1:100 dilution for accurate
        OD). Calculate DNA concentration and proceed to screening step(s) such as Southern blot or
        PCR assay.
    Lysis Buffer                              Genomic DNA Buffer
    50 mM Tris, pH 8.0                        10 mM Tris, pH 8.0
    100 mM EDTA                               0.1 mM EDTA
    100mM NaCl
    1% SDS
    Make both solutions in H20 and filter sterile with 0.2µm Nalgene filter bottle. If SDS precipitates
    warm in 37 C water to re-dissolve.

    Proteinase K stock is 10mg/ml in sterile water. Freeze at –20 C; thaw as needed.
    (Source Roche cat# 1092-766)

								
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