Team:University College London/Protocols/Electrophoresis

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{{:Team:University_College_London/templates/head|coverpicture=week4}}
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<noinclude>{{:Team:University_College_London/templates/head|coverpicture=week4}}
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== Electrophoresis ==
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== Electrophoresis ==</noinclude>
'''Preparing the Gel'''
'''Preparing the Gel'''
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Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water
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'''Step 1:''' Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water
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Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.
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'''Step 2:''' Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.
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Step 3: Cool solution under running cold water.  
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'''Step 3:''' Cool solution under running cold water.  
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Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)
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'''Step 4:''' Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)
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Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles
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'''Step 5:''' Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles
'''Running a gel'''
'''Running a gel'''
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Step 6: Add 1 part loading buffer to five parts of loading sample
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'''Step 6:''' Add 1 part loading buffer to five parts of loading sample
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Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm
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'''Step 7:''' Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm
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Step 8: Add 5ul of DNA ladder to lane 1
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'''Step 8:''' Add 5ul of DNA ladder to lane 1
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Step 9: Add samples to the remaining wells
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'''Step 9:''' Add samples to the remaining wells
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Step 10: Run at 100 volts for 1hour and 15 minutes
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'''Step 10:''' Run at 100 volts for 1hour and 15 minutes
'''Imaging the Gel'''
'''Imaging the Gel'''
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Step 11: Place gel in GelDoc 2000 chamber
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'''Step 11:''' Place gel in GelDoc 2000 chamber
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Step 12: Turn GelDoc 2000 chamber on 
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'''Step 12:''' Turn GelDoc 2000 chamber on 
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Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire
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'''Step 13:''' From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire
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Step 14: Alter the exposure/settings to give a clear image.
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'''Step 14:''' Alter the exposure/settings to give a clear image.
TAE - Tris-acetate-EDTA
TAE - Tris-acetate-EDTA
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EDTA - ethylenediamine tetraacetic acid
EDTA - ethylenediamine tetraacetic acid
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{{:Team:University_College_London/templates/foot}}
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<noinclude>{{:Team:University_College_London/templates/foot}}</noinclude>

Latest revision as of 17:21, 2 August 2012

Electrophoresis

Preparing the Gel

Step 1: Within a conical flask, add 3ml 50X TAE, 1.5g Agarose, and 150ml RO water

Step 2: Heat for 1 min in microwave. Swirl. Heat again for 30s. If solution is clear stop. Else repeat.

Step 3: Cool solution under running cold water.

Step 4: Add 20ul ethidium bromide (normal concentration of EB solution is 500ug/ul)

Step 5: Pour into a sealed casting tray in a slow steady stream, ensuring there are no bubbles

Running a gel

Step 6: Add 1 part loading buffer to five parts of loading sample

Step 7: Position the gel in the tank and add TAE buffer, enough to cover the gel by several mm

Step 8: Add 5ul of DNA ladder to lane 1

Step 9: Add samples to the remaining wells

Step 10: Run at 100 volts for 1hour and 15 minutes

Imaging the Gel

Step 11: Place gel in GelDoc 2000 chamber

Step 12: Turn GelDoc 2000 chamber on 

Step 13: From computer: Quantity One > Scanner > Gel_Doc_Xr>Manuqal Acquire

Step 14: Alter the exposure/settings to give a clear image.

TAE - Tris-acetate-EDTA

EDTA - ethylenediamine tetraacetic acid

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