Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer

From 2010.igem.org

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'''Wear gloves at all times!'''
 
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Requirements:
 
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*30% acrylamide(Biorad) '''NEUROTOXIN!'''
 
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*4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8
 
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*4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol
 
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*TEMED( in yellow cabinet )
 
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*10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge
 
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'''Hoeffer Mighty gel system'''
 
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Casting of the gel:
 
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*Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)
 
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*Clean the plates etc with soap, rinse with demiwater and ethanol, and dry
 
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*Assemble the system in the gel casting holder. Mark the line of separation/stacking
 
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*Mix the separation gel in 10 ml plastic tube:
 
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                              '''for 2 gels'''
 
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'''Seperation gel (0.75 mm)'''              ''' 12.5%'''            '''16%'''   
 
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    30% acrylamide                      4 ml            5.1 ml
 
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    4X separation buffer                2.4 ml          2.4 ml
 
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    MQ                                  3.2 ml          2.1 ml
 
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    10% APS                            28 μl            28 μl
 
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    TEMED                              28 μl            28 μl
 
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*Pipet the separation gel mix immediately in between the glass plates until the marked line is reached
 
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*Pipet water-saturated isobutanol on top of the polymerizing separation gel
 
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*Let the separation gel polymerize completely before preparing the stacking gel
 
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*When the gel is polymerized, discard the isobutanol and wash the gel with water
 
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*Mix the stacking gel in a 10 ml tube:
 
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                                '''for 2 gels'''
 
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    mQ                              1.92 ml
 
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    4X stacking buffer              0.83 ml
 
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    30% acrylamide                  560 μl
 
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    10% APS                          14 μl
 
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    TEMED                            7 μl
 
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*Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker
 
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*Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube
 
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*Disassemble the gel casting holder and take out the gel/plates
 
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*Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C
 
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'''Assembly of the Tris-Glycine gel in the electrophoresis unit'''
 
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*Take the electrophoresis unit of the Hoeffer system and place it next to a power unit
 
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*Dilute 10X and pour 1X electrophoresis buffer in the container( roughly 1 cm high)
 
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*Place gel in the buffer without air bubbles under the gel
 
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*Use the red clamps to place the gel tightly in the unit
 
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*Pour 1X electrophoresis buffer in the chamber so that the top of the gel is immersed
 
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*Take the comb out of the gel and rinse the wells using a hooked needle and syringe
 
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'''Runninge of the gel'''
 
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*Prepare the samples in 1X SDS sample buffer (NOT nucleic acid loading buffer)
 
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*Boil the samples for 5 min and spin down
 
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*Pipet the samples and protein marker carefully into the wells
 
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*Place the electrode cap on the unit and press lightly.Put the other side of the electrode cables in the correct holes of the power unit
 
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*Switch on the power unit and run until the blue front is appr. 1 cm from the end of the gel. Alternatively, use the prestained protein marker to identify the exact point of stopping
 
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*Disassemble the electrophoresis unit and take the gel
 
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*Take one plate off and cut one corner away for positioning purpose
 
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*Carefully bring the gel into a clean staining container using some demi water and/or spacers
 
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*Stain the gel with CBB or silver
 

Latest revision as of 14:11, 24 August 2010