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    Experimental PCR - Notebook Entry
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    • Phusion_PCR_Protocol.pdf
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    Experimental PCR - Notebook Entry

    Monday, 8/17/2020Adapt your own PCR protocol or attach one from the literature or manufacturer.Example PCR Protocol (adapted from Thermo Phusion PCR Kit)Use tables with formulas to make a master mix calculation. Edit the number of reactions to see it in action!PCR Mastermix CalculatorDrag and drop an image from your desktop to view it inline and take notes about it:Gel Electrophoresis Analysis after PCR
    Monday, 8/17/2020
    Adapt your own PCR protocol or attach one from the literature or manufacturer.
    Example PCR Protocol (adapted from Thermo Phusion PCR Kit)
    1.
    Mix into a PCR tube:
    ○
    10 µL Phusion Buffer
    ○
    5 µL 2 mM dNTPs
    ○
    2 µL Forward Primer (10 µM)
    ○
    2 µL Reverse Primer (10 µM)
    ○
    2 µL Genomic Template (less for plasmid template)
    ○
    1 µL Phusion polymerase
    ○
    28 µL Distilled Water (up to 50 µL)
    2.
    PCR Program
    ○
    Start: 98 °C for 30 sec.
    ○
    Cycle: 98 °C for 10 sec.
    ○
    45-72 °C for 15 sec. (use 3 degrees above the primer Tm for annealing temperature)
    ○
    72 °C for 15-30 sec./kb
    ○
    No. of Cycles: 25-35
    ○
    72 °C for 5-10 min.
    ○
    End: 4 °C forever
    Use tables with formulas to make a master mix calculation. Edit the number of reactions to see it in action!
    Drag and drop an image from your desktop to view it inline and take notes about it:
    Gel Electrophoresis Analysis after PCR
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    Lane 1: DNA ladder Lane 2: Template Lane 3: Post-PCR Lane 4: Post-PCR Lane 5: After purification

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