Team:Kyoto/Protocol

From 2012.igem.org

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{{Kyoto/css}}
 
{{Kyoto/header}}
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=Protocol=
=Protocol=
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=====4x Sample buffer=====
=====4x Sample buffer=====
-
  1M Tris-HCl     2mL
+
{| class="wikitable"
-
  SDS             0.8g
+
|1M Tris-HCl
-
  100% glycerol     4mL
+
|2mL
-
  14.7M mercaptoethanol 0.4mL
+
|-
-
  0.5M EDTA       1mL
+
|SDS
-
  Bromophenol blue 8.0mg
+
|0.8g
-
  DW             2.6mL
+
|-
 +
|100% glycerol
 +
|4mL
 +
|-
 +
|14.7M mercaptoethanol
 +
|0.4mL
 +
|-
 +
|0.5M EDTA
 +
|1mL
 +
|-
 +
|Bromophenol blue
 +
|8.0mg
 +
|-
 +
|DW
 +
|2.6mL
 +
|}
 +
 
=====10x electrode buffer=====
=====10x electrode buffer=====
-
  Tris       15.15g
+
{| class="wikitable"
-
  Glycin     71.55g
+
|Tris
-
  10%SDS   50mL
+
|15.15g
-
  DW       450mL
+
|-
-
  Total     500mL
+
|Glycin
 +
|71.55g
 +
|-
 +
|10%SDS
 +
|50mL
 +
|-
 +
|DW
 +
|450mL
 +
|-
 +
|Total
 +
|500mL
 +
|}
 +
 
=====blotting buffer=====
=====blotting buffer=====
-
  Tris       12g
+
{| class="wikitable"
-
  Glycin   14.4g
+
|Tris
-
  DW       800mL
+
|12g
-
  Methanol 200mL
+
|-
 +
|Glycin
 +
|14.4g
 +
|-
 +
|DW
 +
|800mL
 +
|-
 +
|Methanol
 +
|200mL
 +
|}
 +
 
=====TBST=====
=====TBST=====
-
  50mM     Tris
+
{| class="wikitable"
-
  150mM   NaCl
+
|50mM
-
  0.1%     Tween-20
+
|Tris
 +
|-
 +
|150mM
 +
|NaCl
 +
|-
 +
|0.1%
 +
|Tween-20
 +
|}
 +
 
=====blocking buffer=====
=====blocking buffer=====
-
  TBST
+
*TBST
-
  5%  skim milk
+
*5%  skim milk
 +
 
=====AP color development buffer=====
=====AP color development buffer=====
-
  100mM  Tris (pH8.5)
+
*100mM  Tris (pH8.5)
-
  100mM  NaCl
+
*100mM  NaCl
-
  5mM    MgCl2
+
*5mM    MgCl2
-
1. Spin down 100&micro;L culture and suspend into sample buffer.<br>
+
#Spin down 100&micro;L culture and suspend into sample buffer.<br>
-
2. Boil at 95°C for 10 min.<br>
+
#Boil at 95°C for 10 min.<br>
-
3. Apply 10&micro;L to polyacrylamide gel.<br>
+
#Apply 10&micro;L to polyacrylamide gel.<br>
-
4. Electrophorese at 500V, 30mA for 50 min.<br>
+
#Electrophorese at 500V, 30mA for 50 min.<br>
-
5. Transfer at 50V, 100mA for 30 min.<br>
+
#Transfer at 50V, 100mA for 30 min.<br>
-
6. Add blocking buffer to the membrane and incubate with shaking for 30 min. at room temperature.<br>
+
#Add blocking buffer to the membrane and incubate with shaking for 30 min. at room temperature.<br>
-
7. Add appropriate primary antibody diluted in TBST and incubate with shaking for 30 min. at RT.<br>
+
#Add appropriate primary antibody diluted in TBST and incubate with shaking for 30 min. at RT.<br>
-
8. Wash with TBST and incubate with shaking for 10 min, two times.<br>
+
#Wash with TBST and incubate with shaking for 10 min, two times.<br>
-
9. Add secondary antibody conjugated with AP and incubate with shaking for 30 min. at RT.<br>
+
#Add secondary antibody conjugated with AP and incubate with shaking for 30 min. at RT.<br>
-
10. Wash with TBST and incubate with shaking for 10 min, three times.<br>
+
#Wash with TBST and incubate with shaking for 10 min, three times.<br>
-
11. Add 66&micro;L NBT and 33&micro;Lof BCIP into color development buffer., and add the mixture to the membrane.<br>
+
#Add 66&micro;L NBT and 33&micro;Lof BCIP into color development buffer., and add the mixture to the membrane.<br>
-
12.After the coloring, wash with DW.
+
#After the coloring, wash with DW.
 +
===Verification of R9 function by GFP===
 +
'''Verification of R9 function'''  <small>by TAKEUCHI</small><br>
 +
{|class="wikitable"
 +
|R9(20µg/µL)||0.9µL
 +
|-
 +
|GFP(1.2mg/mL)||2.23µL
 +
|-
 +
|RBS||16.85µL
 +
|-
 +
|total||20µL
 +
|}
 +
X5<br>
 +
Method:<br>
 +
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)<br>
 +
2. Put plant cells into GFP&R9 or GFP for 5~30min.<br>
 +
3. Put plant cells into PBS.<br>
 +
4. Hoechst dyeing.<br>
 +
 +
{|class="wikitable"
 +
|||1||2||3||4||5||6
 +
|-
 +
|R9||o||o||o||x||o||o
 +
|-
 +
|cuticle||o||o||o||o||x||x
 +
|-
 +
|soak in GFP||5min||15min||30min||5min||5min||30min
 +
|}
{{Kyoto/footer}}
{{Kyoto/footer}}

Latest revision as of 04:38, 24 September 2012

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Contents

Protocol

Western blotting

Gel solution
Running gelStacking gel
1.5M Tris-HCl(pH8.8)2.5mL-
0.25M Tris-HCl(pH6.8)-3.0mL
30% Acrylamide5mL0.6mL
10% SDS0.2mL0.12mL
DW2.3mL2.3mL
TEMED15µL7µL
10% APS100µL60µL
Total10mL6mL
4x Sample buffer
1M Tris-HCl 2mL
SDS 0.8g
100% glycerol 4mL
14.7M mercaptoethanol 0.4mL
0.5M EDTA 1mL
Bromophenol blue 8.0mg
DW 2.6mL
10x electrode buffer
Tris 15.15g
Glycin 71.55g
10%SDS 50mL
DW 450mL
Total 500mL
blotting buffer
Tris 12g
Glycin 14.4g
DW 800mL
Methanol 200mL
TBST
50mM Tris
150mM NaCl
0.1% Tween-20
blocking buffer
  • TBST
  • 5% skim milk
AP color development buffer
  • 100mM Tris (pH8.5)
  • 100mM NaCl
  • 5mM MgCl2
  1. Spin down 100µL culture and suspend into sample buffer.
  2. Boil at 95°C for 10 min.
  3. Apply 10µL to polyacrylamide gel.
  4. Electrophorese at 500V, 30mA for 50 min.
  5. Transfer at 50V, 100mA for 30 min.
  6. Add blocking buffer to the membrane and incubate with shaking for 30 min. at room temperature.
  7. Add appropriate primary antibody diluted in TBST and incubate with shaking for 30 min. at RT.
  8. Wash with TBST and incubate with shaking for 10 min, two times.
  9. Add secondary antibody conjugated with AP and incubate with shaking for 30 min. at RT.
  10. Wash with TBST and incubate with shaking for 10 min, three times.
  11. Add 66µL NBT and 33µLof BCIP into color development buffer., and add the mixture to the membrane.
  12. After the coloring, wash with DW.

Verification of R9 function by GFP

Verification of R9 function by TAKEUCHI

R9(20µg/µL)0.9µL
GFP(1.2mg/mL)2.23µL
RBS16.85µL
total20µL

X5

Method:
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)
2. Put plant cells into GFP&R9 or GFP for 5~30min.
3. Put plant cells into PBS.
4. Hoechst dyeing.

123456
R9oooxoo
cuticleooooxx
soak in GFP5min15min30min5min5min30min
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