Team:HokkaidoU Japan/Notebook/August16

From 2010.igem.org

(Difference between revisions)
(Restriction Enzyme Digestion)
 
(14 intermediate revisions not shown)
Line 38: Line 38:
!Amount
!Amount
|-
|-
-
|1-1A
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]]
|1 uL
|1 uL
|-
|-
Line 58: Line 58:
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
===コントロール===
+
===Control===
-
* 1 uLのpUC119に1 uLの6x Sample Bufferを加えた
+
* Added 1 uL of 6x Sample Buffer to 1 uL of pUC119
-
* 1 uLの1-1Aに1 uLの6x Sample Bufferを加えた
+
* Also added 1 uL of 6x Sample Buffer to 1 uL of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]]
-
→37℃,60 min
+
** Incubated at 37C for 60 min
 +
 
 +
==Electrophoresis==
 +
 
 +
[[Image:HokkaidoU Japan 20100816a.jpg‎|200px|right|thumb|Electrophoresis of digested DNA]]
 +
 
 +
Added 2 uL of 6x Sample Buffer to digested solution and performed electrophoresis<br>
-
==電気泳動==
 
-
[[Image:HokkaidoU Japan 20100816a.jpg‎|200px|right|thumb|]]
 
-
制限酵素処理した溶液に2 uLの6x Sample Bufferを加え,電気泳動した<br>
 
{|class="protocol"
{|class="protocol"
|-
|-
Line 71: Line 74:
!DNA
!DNA
|-
|-
-
|
+
|1
-
|λ/''Hin''dIII
+
|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII]
|-
|-
-
|
+
|2
-
|pUC119 コントロール
+
|pUC119 control
|-
|-
-
|
+
|3
-
|1-1A コントロール
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]] control
|-
|-
-
|
+
|4
|pUC119 + EcoR I
|pUC119 + EcoR I
|-
|-
-
|
+
|5
|pUC119 + Xba I
|pUC119 + Xba I
|-
|-
-
|
+
|6
-
|1-1A + Spe I
+
|[Team:HokkaidoU_Japan/Parts#BioBricks|[1-1A]] + Spe I
|-
|-
-
|
+
|7
|pUC119 + Pst I
|pUC119 + Pst I
|-
|-
-
|
+
|8
-
|空き
+
|Empty
|}
|}
-
=ベクターの制限酵素処理 (EcoR I, Pst I)=
+
=Restriction Enzyme (EcoR I, Pst I) Digestion of the vector=
 +
 
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
|-
|-
Line 124: Line 128:
|}
|}
-
=パーツの制限酵素処理 (Xba I, Pst I)=
+
=Restriction Enzyme (Xba I, Pst I) Digestion of the Parts=
<div style="float:left;">
<div style="float:left;">
'''RBS'''
'''RBS'''
Line 132: Line 136:
!Amount
!Amount
|-
|-
-
|1-2M
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]
|2.5 uL
|2.5 uL
|-
|-
Line 154: Line 158:
|}
|}
</div>
</div>
-
<div style="float:left;">
+
<div style="float:left; margin-left:50px;">
'''Terminator'''
'''Terminator'''
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
Line 161: Line 165:
!Amount
!Amount
|-
|-
-
|1-23L
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
|1.5 uL
|1.5 uL
|-
|-
Line 185: Line 189:
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
=パーツの制限酵素処理 (EcoR I, Spe I)=
+
=Restriction Enzyme (EcoR I, Spe I) Digestion of the Parts=
<div style="float:left;">
<div style="float:left;">
'''Heat shock promotor'''
'''Heat shock promotor'''
Line 193: Line 197:
!Amount
!Amount
|-
|-
-
|3-1E
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
|5 uL
|5 uL
|-
|-
Line 215: Line 219:
|}
|}
</div>
</div>
-
<div style="float:left;">
+
<div style="float:left; margin-left:50px;">
'''RFP'''
'''RFP'''
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
Line 222: Line 226:
!Amount
!Amount
|-
|-
-
|1-18F
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]]
|5 uL
|5 uL
|-
|-
Line 245: Line 249:
</div>
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
* 制限酵素の活性チェックでSpe Iの活性が低かったので,あとから0.7 uL加えた
+
* It was obvious after restriction enzyme activity check that Spe I was week, so additional 0.7 uL was added

Latest revision as of 07:31, 27 October 2010

Restriction Enzyme Activity Check

Restriction Enzyme Digestion

EcoR I, Xba I, Pst I

Reagent Amount
pUC119 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • Made solution for each EcoR I, Xba I, Pst I
    • incubated at 37C for 60 min

Spe I

Reagent Amount
1-1A 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
    • incubated at 37C for 60 min

Control

  • Added 1 uL of 6x Sample Buffer to 1 uL of pUC119
  • Also added 1 uL of 6x Sample Buffer to 1 uL of 1-1A
    • Incubated at 37C for 60 min

Electrophoresis

Electrophoresis of digested DNA

Added 2 uL of 6x Sample Buffer to digested solution and performed electrophoresis

Lane DNA
1 λ/HindIII
2 pUC119 control
3 1-1A control
4 pUC119 + EcoR I
5 pUC119 + Xba I
6 [1-1A]] + Spe I
7 pUC119 + Pst I
8 Empty

Restriction Enzyme (EcoR I, Pst I) Digestion of the vector

Reagent Amount
pSB1C3 4 uL
DW 10 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Pst I 1 uL
Total 20 uL

Restriction Enzyme (Xba I, Pst I) Digestion of the Parts

RBS

Reagent Amount
1-2M 2.5 uL
DW 11.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

Terminator

Reagent Amount
1-23L 1.5 uL
DW 12.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

Restriction Enzyme (EcoR I, Spe I) Digestion of the Parts

Heat shock promotor

Reagent Amount
3-1E 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL

RFP

Reagent Amount
1-18F 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL
  • It was obvious after restriction enzyme activity check that Spe I was week, so additional 0.7 uL was added