2.4 Testing PCR

Aliquot enough for each pair, plus 2

Materials:

  • Ice bucket with ice

  • 0.2 mL PCR tubes and caps

  • 1 mL Sterile ddH2O (sterile double distilled water)

  • 1.5 mL T10E1 buffer (students already have some, but have more ready)

  • 150 µL dNTP mix (2.5 mM each dNTP) (use up old aliquots)

  • 150 µL 10× polymerase buffer (use up old aliquots) (not Taq buffer)

  • loading dye (at least 75 µL)

  • Low MW Fermentas MassRuler (at least 50 µL)

  • Fill carboy with 1 x TAE

  • Diaper paper

  • Primers

Taq

  • Students can take their 9 μL from the stock bottle, kept on ice at all times
    (4 DNA conc’s @2 µL + ½ rxn worth for pipetting slop)
    Students need Taq at 2U/ μL
    Stock comes as 5U/ μL, so make 100 μL:
    40 μL of 5U/ μL + 60 μL Taq dilution buffer (in freezer)

Make 2 tubes, so both instructors can carry them around in the freezer box

Waterbath at 65C

Gels (1.5%)

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383h-tuth-2015f.xls23.5 KB