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        Primary Amplification of Genomic DNA using DOP - PCR

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        999

        Reagents
        Agarose, Ultrapure
        Gibco, BRL, Cat. no. 15510-027
        10X Buffer, and
        Perkin Elmer (Part no. N808-0010)
        Template DNA
        Use 50-100 ng for each reaction
        Ethidium Bromide
        Research Genetics, Cat. no. 750007
        Loading buffer, 5X
        Quality Biological, Cat. no. 51-026-030
        MgCl2, 25 mM
        Perkin Elmer (Part no. N808-0010)
        100 mM dNTP nucleotides
        dGTP
        Boehringer Mannheim, 1051466
        dCTP
        Boehringer Mannheim, 1051458
        dATP
        Boehringer Mannheim, 1051440
        dTTP
        Boehringer Mannheim, 1051482
        Primer “UN1”
        Midland Certified Reagent Co. Telenius 6MW
        [5’-CCGACTCGAGNNNNNNATGTGG-3’]
        Taq DNA polymerase, 5 U/μl
        Fermentas Life Sciences, Cat. Epo282 500U
        TAE buffer, 10X
        Advanced Biotechnologies, Cat. no.08-514-001
        Sterile water (H20)
        Molecular grade sterile distilled water

        Materials and Equipment
        PCR Thermocycler
        Gel system and power source
        PCR tubes
        Preparation
        1X TAE buffer
        Dilute the 10X TAE with dH20, 1:10 to make 1X TAE
        Dissolve 1 g of agarose in100 ml 1X TAE buffer by warming the solution
        Stock dNTP 2mM
        μl mM final
        dGTP 10 0.2
        dCTP 10 0.2
        dATP 10 0.2
        dTTP 10 0.2
        dH20 460 -
        total 500
        • Autoclave PCR microcentrifuge tubes (0.5 ml size), 2 ml
        microcentrifuge tubes, and pipet tips (10, 200, and 1000 μl).
        • Sterilize pipettes (using UV) to be used for PCR and only use that set
        for PCR (can use Stratalinker or UV light source from tissue culture
        hood).
        • Before starting, make sure that the work space is cleaned with ethanol
        and that it is located in a low traffic area. Use the tissue culture hood
        to maintain sterility if you cannot find a corner to work in.
        Procedure
        1. Label each 0.5 ml tube, being careful to only handle the outside of the tube.
        2. Add appropriate volume of DNA to each PCR tube, close, and set aside into
        ice or 4°C until the reaction mix is ready to aliquot

        3. The order for pipetting the reaction mix is as follows: dH20, buffer, MgCl2,
        dNTP, and primer. The reaction mix for one reaction is as follows (multiply
        each by the number of reactions):
        Primer 4 μl
        Genomic DNA Y μl (Note 1)
        10X buffer 10 μl
        MgCl2 25mM 8 μl
        dNTP 10 μl
        Sterile dH20 X μl (Note 2)
        4. Pipette 96 μl of the reaction mix into each PCR tube (change tips between
        each tube) and put on ice.
        5. Vortex the tubes, spin quickly (few seconds, not higher than 5000 rpm), and
        place on ice. At this point take out the Taq enzyme, mix carefully (tap with
        finger), spin quickly, then add 1 μl (for each 100 μl) (Note 3) to each reaction.
        Vortex the tubes, spin quickly, and put back on ice.
        6. Vortex each tube, spin quickly, and put them all into the PCR machine and
        run using the appropriate PCR program (Note 4).
        7. After completion, remove the tubes from the PCR machine, vortex, spin
        quickly, and place on ice. Mix 0.8 μl of 5X DNA loading buffer with a 2 μl
        aliquot from each reaction to run on a 1% agarose gel. The resulting smear
        migrates around 500 bps (Note 5).
        Notes
        1. The volume of genomic DNA depends on the concentration that you have
        available.
        2. The volume of water to add depends on the volume of DNA that is added, and
        the total reaction volume should be equal to 100 μl.
        3. The Taq polymerase should always be added last, and since it is more viscous
        and sticky, it needs to be mixed well before each use. Use the pipet tip you
        are about to draw with to gently stir the contents as you draw up the enzyme.

        4. PCR program (runs approximately 7 hours):
        Step Temperature (°C) Minutes
        1 (initial denaturation). 93 10
        2 94 1
        3 30 1.5
        4 ramp 30-70 3
        5 72 3
        6 repeat steps 2-5, 4 times
        7 94 1
        8 62 1
        9 72 3 + 1 second/cycle
        10 repeat steps 7-9, 34 times
        11 72 10
        12 4 ∞
        5. 1% agarose gel

        <center> <p>  </p> </center>
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