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        Vectorette PCR of Yeast DNA

        互联网

        1049

         

        Vectorette PCR of Yeast DNA

        Carl Friddle

        1) Cut 1-3 µg of clean DNA overnight with 8-10U of blunt cutting enzyme in 20µl

        Most problems come from dirty, uncut DNA. Phenol/glass bead/RNase prepared DNA works well

        RsaI, AluI and DraI provide good results.

        2) Heat inactivate enzyme

        3) Add:

        • 3µl 10x NEBuffer used in digest
        • 1µl annealed anchor bubble
        • 1µl (400U) ligase
        • 0.5µl of 5mM ATP (50µM ATP final)
        • 25.5µl Water
        4) Incubate at 16 C for 9-24 hours.

        5) Use 5µl in 100µl PCR. Perkin Elmer Ampliwax is recommended for hot start.

        • 5 µl of ligation
        • 2.5 µl of 20µM specific primer [M13(-47) for mTn3 library]
        • 2.5 µl of 20µM 224 primer
        • 8 µl of 2.5 mM dNTPs
        • 10 µl of Taq PCR buffer
        • 71µl Water
        • 1µl Taq DNA polymerase (5U)
        • Transfer to Perkin Elmer 9600 Thermal Cycler
          • Denature 92C, 2 minutes
          • 35 Cycles [92C, 20sec; 67C, 30sec; 72C, 45-180sec (>1 min/1 kb)]
          • 72C, 90sec
        6) Gel purify 80 µl of PCR product in 1-3% SeaKem GTG, extract with Qiaex (Qiagen), elute with 12 µl of ddWater

        7) Sequence 7 µl with Sequenase kit from Amersham.

        Use 1 µl of 200-600µM specific primer [M13(-47) for mTn3]. Undiluted 10 OD synthesis from Genset works well.

        Use high specific activity S-35 (>1000 Ci/mmol, Amersham AG1000)

        Boil 10' and fast cool in ice water.

         


        Anchor Bubble primers
        3'   GAGAGGGAAGAGAGCAGGCAAGGAATGGAAGCTGTCTGTCGCAGGAGAGGAAG 5'
             ||||||||||||   ||    || | | | ||| |  |   ||||||||||||
        5' GACTCTCCCTTCTCGAATCGTAACCGTTCGTACGAGAATCGCTGTCCTCTCCTTC  3'
        
        
        PRIMER 224   5' CGAATCGTAACCGTTCGTACGAGAATCGCT 3'
        
        
        

        To anneal bubble primers, heat a 2-4µM (in ddWater) to 65 C for 5 minutes, then add MgCl2 to 1-2 mM and allow to cool to room temperature.

        Would you like to see a diagram ?

         


        References:

        • Riley et al, Nucleic Acids Research 18: 2887-2890, 1990
        • Foote et al, Science 258: 60-66, 1992
        • Burns et al, Genes Dev. 8(9): 1087-1105, 1994
        • Vollrath, Large DNA Course, Cold Spring Harbor Laboratory, 1995

         

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