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        Mouse p27 PCR Using Gitschier Buffer

        互联网

        1634

         

        Mutant allele: Neo-1 primer (CCTTCTATCGCCTTCTTG), plus mgK-3 primer (TGGAACCCTGTGCCATCTCTAT) produce a 0.5kB PCR product.

        Wildtype allele: mgK-3 (above) plus mcK-5 (GAGCAGACGCCCAAGAAGC) produce ~1kB product.

        Stock Solutions:

        1 M Tris pH 8.8 (do not use pH meter, store at room temp.)
        1.23 g Tris HCl
        5.13 g Tris base
        q.s. 50 mL with H2 O

        KG-1 (10x) (Store frozen)

        Amt   [final]
        8.3 mL 1M (NH4 )2 SO4 166mM
        33.5 mL 1 M Tris base pH 8.8 670 mM
        174 µL ß -Mercaptoethanol 50 mM
        3.35 mL 1M MgCl 2 67 mM
        q.s. to 50 mL with H2O and aliquot into 1.5 mL eppendorf tubes

         


        PCR Reaction Mix (To make a fresh master mix, multiply # PCR reactions x volumes, below)

        2 µL KG-1 (10x) 1x
        2 µL KG-2 (10x) 1x
        2 µL Primer 1 (1uM) 0.1 mM
        2 µL Primer 2 (1uM) 0.1 mM
        0.2 µL Taq (Gibco) (5 U/uL) 0.05 U/mL
        10 µL H2O
        1.8 µL DNA
        20 µL Total

         

        Cycling Parameters: It is important to not place the tubes on the machine until the block has heated to > 90ºC.  The first 4 cycles employ a higher melting temperature which helps to denature genomic DNA.  Subsequent cycles use a lower melt which is sufficient for PCR product and preserves enzyme function.  Longer (2 min.) extension times should be used if products > 2 kb are being amplified.

        95°C hold x 2 min.  (while inserting tubes)
        (96°C x 30", 57°C x 30", 65°C x 1-2') x4
        (93°C x 30", 57°C x 30", 65°C x 1-2') x36
        4°C hold

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