Team:UNIPV-Pavia/Protocols/Pcr

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*[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]]
*[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]]
*[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]]
*[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]]
 +
*[[Team:UNIPV-Pavia/Protocols/Precipitation|DNA precipitation with sodium acetate]]
*[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]]
*[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]]
*[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]]
*[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]]
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<h1>Antarctic Phosphatase</h1>
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<h1>PCR</h1>
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''(estimated time: 1 hour and 30 min)''
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''(estimated time: 3 hours and 30 min)''
<br>
<br>
<br>
<br>
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**0.6 µl MgCl2
**0.6 µl MgCl2
**0.4 µl dNTPs
**0.4 µl dNTPs
-
**1 µl DNA (or ddH2O for blank sample)
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**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
**0.2 µl Taq Polymerase
**250 nM VF2 primer
**250 nM VF2 primer
**250 nM VR primer
**250 nM VR primer
**A proper amount of ddH2O to have 20 µl of total reaction volume
**A proper amount of ddH2O to have 20 µl of total reaction volume
-
*into a  
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*into an eppendorf tube.
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*Put the eppendorf tube in the thermal cycler and set this program:
 +
**95°C 10 min
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**CYCLE:
 +
***95°C 30 sec
 +
***60°C 1 min
 +
***72°C 1-3 min
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**for 35 cycles
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**72°C 7 min
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**16°C forever.
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*Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.
<br>
<br>

Latest revision as of 10:16, 27 July 2008

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The protocols we used


PCR

(estimated time: 3 hours and 30 min)

Materials needed:

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


  • 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.