Team:EPF-Lausanne/Protocols/SDS-PAGE

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<font size="6" color="#007CBC">PCR with <i>Taq Platinium</i> Protocol</font>  
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<font size="6" color="#007CBC">SDS-PAGE protocol</font>  
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<font size="3">Protein purification</font>
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Lysis Buffer (2x) : for 100 ml
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*4g of SDS
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*20ml of glycerol
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*2ml of 2-Mercaptoethanol
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*70ml of stacking buffer (0.5 M Tris-HCl, pH=6.8)
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*100mg of Bromophenol blue
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*8ml of dd water
    
    
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'''1.''' Add the following components to a sterile 0.5-ml microcentrifuge tube. Volumes are for a single 50-μl reaction, and can be scaled as needed. Prepare a master mix of common components for multiple reactions.
 
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{| class="wikitable" style="text-align:center; width:60%;"
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! scope=col | Components
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! scope=col | Volume
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! scope=col | Final concentration
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|10X PCR Buffer, Minus Mg
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|2,5 μl
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|1X
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|10 mM dNTP mixture
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|1 μl
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|0.2 mM each
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|50 mM MgCl2
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|1.5 μl
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|1.5 mM
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|Primer mix (10 μM each)
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|1 μl
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|0.2 μM each
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|Template DNA
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|≥1 μl
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|(as required)
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|Platinum® Taq DNA Polymerase
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|0.2 μl 
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|1.0 unit*
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|Autoclaved, distilled water 
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|to 25 μl
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|Not applicable
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'''*'''1.0 unit is sufficient for amplifying most targets. In some cases, more enzyme may be required (up to 2.5 units).
 
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'''2.''' Cap the tubes, mix, and centrifuge briefly to collect the contents.
 
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'''3.''' Incubate tubes in a thermal cycler at 94°C for 30 seconds to 2 minutes to completely denature the template and activate the enzyme.
 
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'''4.''' Perform 25–35 cycles of PCR amplification as follows:
 
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! scope=row | Denature
 
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| 94°C for 30 seconds
 
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! scope=row | Anneal
 
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|55°C for 30 seconds
 
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! scope=row | Extend
 
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|72°C for 1 minutes per kb
 
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'''5.''' Maintain the reaction at 4°C after cycling.  The samples can be stored at –20°C until use.
 
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Analyze the products by agarose gel electrophoresis.
 
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Revision as of 12:55, 21 October 2009



SDS-PAGE protocol

Protein purification
Lysis Buffer (2x) : for 100 ml

  • 4g of SDS
  • 20ml of glycerol
  • 2ml of 2-Mercaptoethanol
  • 70ml of stacking buffer (0.5 M Tris-HCl, pH=6.8)
  • 100mg of Bromophenol blue
  • 8ml of dd water


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