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| | =General Protocol for Light App Experiments= | | =General Protocol for Light App Experiments= |
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| | ==Overnight cultures== | | ==Overnight cultures== |
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| | #Cast a gel | | #Cast a gel |
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| | '''The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide:''' | | '''The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide:''' |
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| - | {| border="1" cellpadding="5" cellspacing="0" align="center"
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| - | ! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)
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| - | |-=
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| - | | align="center"|0.5 || align="center"|1 - 30
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| - | |-
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| - | | align="center"|0.7 || align="center"|0.8 - 12
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| - | |-
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| - | | align="center"|1.0 || align="center"|0.5 - 10
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| - | |-
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| - | | align="center"|1.2 || align="center"|0.4 - 7
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| - | |-
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| - | | align="center"|1.5 || align="center"|0.2 - 3
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| - | |}
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| - | # Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox).
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| - | # Microwave until the agarose is fully melted. This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results. As long as you do not burn the agarose and nothing bubbles over, this step is robust.
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| - | # '''From here on, a heat protective glove should be used any time the heated flask must be touched!'''
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| - | # Let the agarose cool on the bench for ~5 minutes.
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| - | # At this point add your DNA visualization reagent, e.g., ethidium bromide at a volume appropriate for the amount of melted agarose. This amount will depend on the concentration of the stock solution of the stain.
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| - | # The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles, and should therefore be periodically swirled while the agarose is cooling.
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| - | # Pour the agarose solution into the gel casting apparatus. A pipette tip should be used to pop or shove to the side any bubbles.
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| - | # After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
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| - | # Because removal of the comb too early can cause the wells to collapse on themselves, one should always poor a gel prior to prepping the samples.
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| - | Adapted thanks to [http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].
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| | '''← [[Team:Washington/Protocols|Back to Protocols]]''' | | '''← [[Team:Washington/Protocols|Back to Protocols]]''' |
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