Team:UNIPV-Pavia/Methods Materials/PCR

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**16°C forever.
**16°C forever.
*Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.
*Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.
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Revision as of 12:00, 30 June 2009

EthanolPVanimation.gif



Protocols

PCR

(estimated time: 3 hours and 30 min)

  • For every DNA sample you want to amplify, put:
    • 2 µl buffer
    • 0.6 µl MgCl2
    • 0.4 µl dNTPs
    • 1 µl DNA (or ddH2O for blank sample). If you are performing a colony PCR, pick up the desired colony from a plate with a tip and dip it in the solution.
    • 0.2 µl Taq Polymerase
    • 250 nM VF2 primer
    • 250 nM VR primer
    • A proper amount of ddH2O to have 20 µl of total reaction volume
  • into an eppendorf tube.
  • Put the eppendorf tube in the thermal cycler and set this program:
    • 95°C 10 min
    • CYCLE:
      • 95°C 30 sec
      • 60°C 1 min
      • 72°C 1-3 min
    • for 35 cycles
    • 72°C 7 min
    • 16°C forever.
  • Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.

  • MgCl2
  • Buffer
  • dNTPs
  • ddH2O
  • Taq Polymerase
  • VF2 primer
  • VR primer




Retrieved from "http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/PCR"