Talk:Team:EPF-Lausanne/Protocols

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(Created page with "{{:Team:EPF-Lausanne/Templates/Header|title=Agarose gel electrophoresis}} made on 01.07.2011 == Purpose == To analyse DNA composition of a sample. Voltage-dependent DNA migrat...")
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{{:Team:EPF-Lausanne/Templates/Header|title=Agarose gel electrophoresis}}
{{:Team:EPF-Lausanne/Templates/Header|title=Agarose gel electrophoresis}}
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made on 01.07.2011
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Written on 01.07.2011
== Purpose ==
== Purpose ==
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To analyse DNA composition of a sample. Voltage-dependent DNA migration through agarose gel allows sequences separation according to their size.  
To analyse DNA composition of a sample. Voltage-dependent DNA migration through agarose gel allows sequences separation according to their size.  
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==Ingredients==
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==Ingredients & Materials==
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for a 1% agarose gel
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For a 1% agarose gel
{| class="wikitable" style="text-align:center; width:80%;"
{| class="wikitable" style="text-align:center; width:80%;"
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+Gel size
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|+ Ingredients for different gel cast sizes
|-
|-
! scope=row |  
! scope=row |  
|Small  
|Small  
|Medium
|Medium
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|Big 26 wells
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|Big: 26 wells
|-
|-
! scope=row | Agarose
! scope=row | Agarose
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|2.5µl
|2.5µl
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|-
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! scope=row | Gel Red
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! scope=row | GelRed
|2g
|2g
|200ml
|200ml
|5µl
|5µl
|}
|}
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Sample loading buffer will also be needed.
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Materials: bottle, tray, cast, comb, electrophoresis chamber, voltage/current generator, pipette, microwave, weight, spoon, labcoat...
== Method ==
== Method ==
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<p>•      Weight agarose, place it into a bottle with a cap, add TBE 1x buffer</p>
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* To prepare the gel:
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<p>•      Place the bottle into a microwave, <gras>loosen the cap</gras>, heat it untill it almost boils and the agar is dissolved</p>
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***    Weight agarose, place it into a bottle with a cap, add TBE 1x buffer
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<p>• Cool it down until it can be hold (that can be done under water)</p>
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***  Place the bottle into a microwave, <gras>loosen the cap</gras>, heat it untill it almost boils and the agar is dissolved
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<p>• Add 1.25µl of Gel Red</p>
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***  Cool it down until it can be hold (that can be done under water)
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<p>• Pour into the rack, put the comb and wait while it polymerizes</p>
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***  Add 1.25µl of GelRed
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<p> '' Usually “B” valves are starting to load earlier (leave the button unpressurised prior to the pressure increase, it tends to stick to the slide.)</p>
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***  Pour into the casting tray (it is easy to assemble the casting but a way to difficult to discribe), put the comb and wait until it polymerizes
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<p>  Increase the pressure in both “A” and “B” valves till ~13-15 psi and make sure that you can see that all chambers are separated. If no – increase the pressure''</p>
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* To run the gel:
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** Take out the comb and place the tray with your gel into the electrophoresis chamber (usually already filled with buffer) so that your wells are nearer to the cation, red side.
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**    TBE 1x buffer should be covering the tray, add more if needed
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**    In a PCR tube mix ~10µl of your sample with 1.5µl of loading solution (quantities indicated may vary with the sample concentration )
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**    Load about 5µl of your sample (this quantity varies with the thickness of the comb used and DNA concentration) and DNA ladder onto gel, taking care not to diffuse it and not to displace the gel
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**    Place the cover, red wire to red side and inversely, plug it into the tension/current generator
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**    On the generator set for voltage: constant mode 80V and time: 60min  (faster run with higher voltage as for example 150V may affect the quality of bands separation)
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**    After run is over, take the tray out on some plate, and bring it to visualize DNA bands to a UV lightbox or gel imaging system
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**    Gel can be disposed in a normal biological waste or kept covered in an aluminum foil
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 21:09, 1 July 2011

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