Team:UNIPV-Pavia/Methods Materials/PCR

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(New page: <h1>PCR</h1> ''(estimated time: 3 hours and 30 min)'' <br> <br> '''Materials needed:''' *'''MgCl2''' *'''Buffer''' *'''dNTPs''' *'''ddH2O''' *'''Taq Polymerase''' *'''VF2 primer''' *'''VR...)
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    <font face="Pristina" size="50" ><b>Protocols</b></font>
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=='''PCR'''==
''(estimated time: 3 hours and 30 min)''
''(estimated time: 3 hours and 30 min)''
<br>
<br>
<br>
<br>
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'''Materials needed:'''
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*'''MgCl2'''
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*'''Buffer'''
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*'''dNTPs'''
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*'''ddH2O'''
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*'''Taq Polymerase'''
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*'''VF2 primer'''
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*'''VR primer'''
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<br>
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*For every DNA sample you want to amplify, put:
*For every DNA sample you want to amplify, put:
**2 µl buffer
**2 µl buffer
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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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<tr bgcolor='yellow'><td>INGREDIENTS:
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'''MgCl2'''<hr color='white' width='70%'>
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'''Buffer'''<hr color='white' width='70%'>
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'''dNTPs'''<hr color='white' width='70%'>
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'''ddH2O'''<hr color='white' width='70%'>
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'''Taq Polymerase'''<hr color='white' width='70%'>
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'''VF2 primer'''<hr color='white' width='70%'>
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'''VR primer'''</font>
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Latest revision as of 10:33, 21 October 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.

INGREDIENTS:

MgCl2
Buffer
dNTPs
ddH2O
Taq Polymerase
VF2 primer

VR primer





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