Team:UNIPV-Pavia/Methods Materials/Electrophoresis

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     <font face="Pristina" size="50" ><b>Protocols</b></font>
     <font face="Pristina" size="50" ><b>Protocols</b></font>
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'''Materials needed:'''
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*'''DNA samples'''
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*Prepare agarose gel in 1x TBE buffer
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*'''Ethidium bromide'''
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*'''Loading buffer 10x Blue Juice, Invitrogen'''
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*'''TBE (Tris/Borate/EDTA buffer)'''
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** '''TBE 5x (final volume 1 liter)'''
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**'''54 gr Tris'''
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**'''27.5 gr Borate'''
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**'''20 ml EDTA 0.5 M (pH 8)'''
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*'''Marker (1 kb DNA Ladder, Promega)'''
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*'''Face mask and gloves'''
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*'''Electrophoresis apparatus'''
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*'''Transilluminator'''
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*Prepare 1% agarose gel in 1x TBE buffer
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*Add ethidium bromide (using gloves and face mask for your safety):
*Add ethidium bromide (using gloves and face mask for your safety):
**1 µl in the small size agarose gel (70 ml)
**1 µl in the small size agarose gel (70 ml)
**2 µl in the middle size agarose gel (150 ml)  
**2 µl in the middle size agarose gel (150 ml)  
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**4 µl in the big size agarose gel (220 ml)
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**4 µl in the big size agarose gel (250 ml)
*Cast the gel, insert the well-forming comb and let it polymerize
*Cast the gel, insert the well-forming comb and let it polymerize
*Add the loading buffer to each sample
*Add the loading buffer to each sample
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*Load the samples and 8 µl marker  
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*Load the samples and 8 µl of marker  
*Set to 70-100 volts and electrophorese for the required amount of time
*Set to 70-100 volts and electrophorese for the required amount of time
*Use UV-light to look at the bands
*Use UV-light to look at the bands
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*Take a picture of the gel, if needed (not when bands have to be cut)
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*Take a picture of the gel, if needed (not when bands have to be cut!!!)
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<tr bgcolor='yellow'><td>INGREDIENTS:
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'''DNA samples'''<hr color='white' width='70%'>
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'''Ethidium bromide'''<hr color='white' width='70%'>
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'''Loading buffer 10x Blue Juice, Invitrogen'''<hr color='white' width='70%'>
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'''TBE (Tris/Borate/EDTA buffer) 5x (final volume 1 liter)'''
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*'''54 gr Tris'''
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*'''27.5 gr Borate'''<hr color='white' width='70%'>
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'''20 ml EDTA 0.5 M (pH 8)'''<hr color='white' width='70%'>
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'''Marker (1 kb DNA Ladder, Promega)'''<hr color='white' width='70%'>
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'''Face mask and gloves'''<hr color='white' width='70%'>
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'''Electrophoresis apparatus'''<hr color='white' width='70%'>
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'''Transilluminator'''</font>
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*NOTE: when not specified, the marker has the following pattern:
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{|align="center"
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|[[Image:pv_ladder.jpg]]
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Latest revision as of 10:30, 21 October 2009

EthanolPVanimation.gif



Protocols


Electrophoresis

(estimated time: 2 hours)

  • Prepare agarose gel in 1x TBE buffer
  • Add ethidium bromide (using gloves and face mask for your safety):
    • 1 µl in the small size agarose gel (70 ml)
    • 2 µl in the middle size agarose gel (150 ml)
    • 4 µl in the big size agarose gel (250 ml)
  • Cast the gel, insert the well-forming comb and let it polymerize
  • Add the loading buffer to each sample
  • Load the samples and 8 µl of marker
  • Set to 70-100 volts and electrophorese for the required amount of time
  • Use UV-light to look at the bands
  • Take a picture of the gel, if needed (not when bands have to be cut!!!)










INGREDIENTS:

DNA samples
Ethidium bromide
Loading buffer 10x Blue Juice, Invitrogen

TBE (Tris/Borate/EDTA buffer) 5x (final volume 1 liter)

  • 54 gr Tris
  • 27.5 gr Borate
20 ml EDTA 0.5 M (pH 8)
Marker (1 kb DNA Ladder, Promega)
Face mask and gloves
Electrophoresis apparatus

Transilluminator





  • NOTE: when not specified, the marker has the following pattern:
Pv ladder.jpg
Retrieved from "http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Electrophoresis"