Difference between revisions of "Preparing a SDS-PAGE Gel"

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(Created page with '==Separating Gel== *For 12 mL final volume (2 gels) *Combine, adding APS and TEMED last *Pour into cassette, overlay with the top layer of water saturated butanol {| border="1" !...')
 
(added SOP requirement-JR)
 
(3 intermediate revisions by one other user not shown)
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==SOP==
 +
*[[SOP- Electrophoresis]]
 +
 
==Separating Gel==
 
==Separating Gel==
 
*For 12 mL final volume (2 gels)
 
*For 12 mL final volume (2 gels)
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*Pour into cassette, overlay with the top layer of water saturated butanol
 
*Pour into cassette, overlay with the top layer of water saturated butanol
 
{| border="1"
 
{| border="1"
! Component || 7.5% || 10% || 12.5%
+
! Component || 6% ||7.5% || 10% || 12.5%
 
|-
 
|-
| [[SDS-PAGE Separating Gel Buffer]] || 3 mL || 4 mL || 5 mL
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| [[SDS-PAGE Separating Gel Buffer]]|| 3 mL || 3 mL || 3 mL || 3 mL
 
|-
 
|-
| [[Acrylamide Solution]] || 3 mL || 3 mL || 3 mL
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| [[Acrylamide Solution]]|| 2.4 mL || 3 mL || 4 mL || 5 mL
 
|-  
 
|-  
| Water || 6 mL || 5 mL || 4mL
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| Water || 6.6 mL|| 6 mL || 5 mL || 4mL
 
|-  
 
|-  
| APS (make fresh, 0.1g/mL in water) || 200 uL || 200 uL || 200 uL
+
| APS (make fresh, 0.1g/mL in water) || 200uL || 200 uL || 200 uL || 200 uL
 
|-
 
|-
| TEMED || 30 uL || 30 uL || 30uL
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| TEMED || 30 uL || 30 uL || 30 uL || 30uL
 
|}
 
|}
 +
*For a [[Phos-Tag Electrophoresis and Western Blotting|Phos-Tag Gel]], add 24 uL 5 mM Phos-Tag and 24 uL 10 mM MnCl in the separating gel only
  
 
==Stacking Gel==
 
==Stacking Gel==
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| Water || 4 mL
 
| Water || 4 mL
 
|-  
 
|-  
| APS (make fresh, 0.1g/mL in water) || 200 uL || 200 uL || 200 uL
+
| APS (make fresh, 0.1g/mL in water) || 200 uL  
 
|-
 
|-
| TEMED || 30 uL || 30 uL || 30uL
+
| TEMED || 30 uL  
 
|}
 
|}
  
 
[[Category: SDS-PAGE]]
 
[[Category: SDS-PAGE]]
 
[[Category: Electrophoresis]]
 
[[Category: Electrophoresis]]
 +
[[Category: PhosTag]]
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[[Category: Phosphorylation]]

Latest revision as of 16:38, 4 June 2020

SOP

Separating Gel

  • For 12 mL final volume (2 gels)
  • Combine, adding APS and TEMED last
  • Pour into cassette, overlay with the top layer of water saturated butanol
Component 6% 7.5% 10% 12.5%
SDS-PAGE Separating Gel Buffer 3 mL 3 mL 3 mL 3 mL
Acrylamide Solution 2.4 mL 3 mL 4 mL 5 mL
Water 6.6 mL 6 mL 5 mL 4mL
APS (make fresh, 0.1g/mL in water) 200uL 200 uL 200 uL 200 uL
TEMED 30 uL 30 uL 30 uL 30uL
  • For a Phos-Tag Gel, add 24 uL 5 mM Phos-Tag and 24 uL 10 mM MnCl in the separating gel only

Stacking Gel

  • Rinse polymerized separating gel with water
  • For 10 mL (2 gels)
  • Combine, adding APS and TEMED last
  • Pour over separating gel and insert comb
Component
SDS-PAGE Stacking Gel Buffer 2.5 mL
Acrylamide Solution 1.7 mL
Water 4 mL
APS (make fresh, 0.1g/mL in water) 200 uL
TEMED 30 uL