Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer
From 2010.igem.org
(Difference between revisions)
(New page: '''Wear gloves at all times!''' Requirements: *30% acrylamide(Biorad) '''NEUROTOXIN!''' *4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8 *4X stacking gel buffer: 0.5M Tris.HCl, 1...) |
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*10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge | *10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge | ||
- | Hoeffer Mighty gel system | + | '''Hoeffer Mighty gel system''' |
+ | Casting of the gel: | ||
+ | *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) | ||
+ | *Clean the plates etc with soap, rinse with demiwater and ethanol, and dry | ||
+ | *Assemble the system in the gel casting holder. Mark the line of separation/stacking | ||
+ | *Mix the separation gel in 10 ml plastic tube: | ||
+ | '''for 2 gels''' | ||
+ | '''Seperation gel (0.75 mm)''' ''' 12.5%''' '''16%''' | ||
+ | 30% acrylamide 4 ml 5.1 ml | ||
+ | 4X separation buffer 2.4 ml 2.4 ml | ||
+ | MQ 3.2 ml 2.1 ml | ||
+ | 10% APS 28 μl 28 μl | ||
+ | TEMED 28 μl 28 μl | ||
+ | *Pipet the separation gel mix immediately in between the glass plates until the marked line is reached | ||
+ | *Pipet water-saturated isobutanol on top of the polymerizing separation gel | ||
+ | *Let the separation gel polymerize completely before preparing the stacking gel | ||
+ | *When the gel is polymerized, discard the isobutanol and wash the gel with water | ||
+ | *Mix the stacking gel in a 10 ml tube: | ||
+ | '''for 2 gels''' | ||
+ | mQ 1.92 ml | ||
+ | 4X stacking buffer 0.83 ml | ||
+ | 30% acrylamide 560 μl | ||
+ | 10% APS 14 μl | ||
+ | TEMED 7 μl | ||
+ | *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 | ||
+ | *Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube | ||
+ | *Disassemble the gel casting holder and take out the gel/plates | ||
+ | *Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C | ||
+ | |||
+ | '''Assembly of the Tris-Glycine gel in the electrophoresis unit''' |
Revision as of 13:48, 24 August 2010
Wear gloves at all times!
Requirements:
- 30% acrylamide(Biorad) NEUROTOXIN!
- 4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8
- 4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol
- TEMED( in yellow cabinet )
- 10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge
Hoeffer Mighty gel system Casting of the gel:
- 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)
- Clean the plates etc with soap, rinse with demiwater and ethanol, and dry
- Assemble the system in the gel casting holder. Mark the line of separation/stacking
- Mix the separation gel in 10 ml plastic tube:
for 2 gels Seperation gel (0.75 mm) 12.5% 16% 30% acrylamide 4 ml 5.1 ml 4X separation buffer 2.4 ml 2.4 ml MQ 3.2 ml 2.1 ml 10% APS 28 μl 28 μl TEMED 28 μl 28 μl
- Pipet the separation gel mix immediately in between the glass plates until the marked line is reached
- Pipet water-saturated isobutanol on top of the polymerizing separation gel
- Let the separation gel polymerize completely before preparing the stacking gel
- When the gel is polymerized, discard the isobutanol and wash the gel with water
- Mix the stacking gel in a 10 ml tube:
for 2 gels mQ 1.92 ml 4X stacking buffer 0.83 ml 30% acrylamide 560 μl 10% APS 14 μl TEMED 7 μl
- 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
- Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube
- Disassemble the gel casting holder and take out the gel/plates
- Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C
Assembly of the Tris-Glycine gel in the electrophoresis unit