Team:HokkaidoU Japan/Notebook/September17

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September17|September 17]]</div></div>
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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September20|September 20]]</div></div>
*Construction of GFP marker for a part which will be secreted using T3SS
*Construction of GFP marker for a part which will be secreted using T3SS
*Ordered primers for construction for same part
*Ordered primers for construction for same part
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<!--      Description -->|GFP
<!--      Description -->|GFP
<!--      BioBrick No. -->|BBa_E0040
<!--      BioBrick No. -->|BBa_E0040
-
<!--          Well No. -->|1-14K
+
<!--          Well No. -->|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]]
<!--            Length -->|720bp
<!--            Length -->|720bp
<!--          Plasmid -->|pSB1A3
<!--          Plasmid -->|pSB1A3
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<!--      Description -->|double terminator
<!--      Description -->|double terminator
<!--      BioBrick No. -->|BBa_B0015
<!--      BioBrick No. -->|BBa_B0015
-
<!--          Well No. -->|1-23L
+
<!--          Well No. -->|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
<!--            Length -->|129bp
<!--            Length -->|129bp
<!--          Plasmid -->|pSB1AK3
<!--          Plasmid -->|pSB1AK3
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* Performed electrophoresis of 1-14K and 1-23L to estimate concentration of each solution.
+
* Performed electrophoresis of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]] and [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] to estimate concentration of each solution.
* Estimated concentration from photo of electrophoresis  
* Estimated concentration from photo of electrophoresis  
* pSB1A3 solution was done by other person.
* pSB1A3 solution was done by other person.
* Made digestion recipes(below) based on estimated concentrations
* Made digestion recipes(below) based on estimated concentrations
-
* Made 30ul of pSB1A3 solution, but latter found it insufficient to ligate parts
+
* Made 30 ul of pSB1A3 solution, but latter found it insufficient to ligate parts
** made more 50ul of it after
** made more 50ul of it after
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!Amount
!Amount
|-
|-
-
|1-14K
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]]
|200 ng/ul
|200 ng/ul
|-
|-
-
|1-23L
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
|120 ng/ul
|120 ng/ul
|-
|-
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!Amount
!Amount
|-
|-
-
|1-23L
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
|0.5 uL
|0.5 uL
|-
|-
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|35 uL
|35 uL
|-
|-
-
|style="border-top:1px solid #000;"|'''Total'''
+
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''50 uL'''
+
|style="border-top:1px solid #996;"|'''50 uL'''
|}
|}
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!Amount
!Amount
|-
|-
-
|1-14K
+
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]]
|1.5 uL  
|1.5 uL  
|-
|-
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|13 uL
|13 uL
|-
|-
-
|style="border-top:1px solid #000;"|'''Total'''
+
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''20 uL'''
+
|style="border-top:1px solid #996;"|'''20 uL'''
|}
|}
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|3 uL
|3 uL
|-
|-
-
|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''30 uL'''
+
|style="border-top:1px solid #996;"|'''30 uL'''
|}
|}
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|9 uL
|9 uL
|-
|-
-
|style="border-top:1px solid #000;"|'''Total'''
+
|style="border-top:1px solid #996;"|'''Total'''
-
|style="border-top:1px solid #000;"|'''30 uL'''
+
|style="border-top:1px solid #996;"|'''30 uL'''
|}
|}
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* Incubated each solution at 37C
* Incubated each solution at 37C
-
* Solution of 1-23L was incubated for 150 min
+
* Solution of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] was incubated for 150 min
-
* Solution of 1-14K was incubated for 90 min
+
* Solution of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]] was incubated for 90 min
-
* 30ul of pSB1A3 solution was incubated for 60 min
+
* 30 ul of pSB1A3 solution was incubated for 60 min
-
* 50ul of pSB1A3 solution was incubated for 30 min
+
* 50 ul of pSB1A3 solution was incubated for 30 min
* Performed electrophoresis for each solution
* Performed electrophoresis for each solution
-
* put 12uls each into wells of a gel like below.
+
* put 12 uls each into wells of a gel like below.
{|class="protocol"
{|class="protocol"
 +
|'''Lane'''
 +
|'''DNA'''
|-
|-
|1
|1
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'''Results'''
'''Results'''
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* Could not see bands of 1-23L because leaked out
+
* Could not see bands of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] because leaked out
** Drove current for too long
** Drove current for too long
* Extracted the other samples from a gel using promega kit
* Extracted the other samples from a gel using promega kit
* Stored all at -20C.
* Stored all at -20C.

Latest revision as of 08:27, 27 October 2010

Notebook
  • Construction of GFP marker for a part which will be secreted using T3SS
  • Ordered primers for construction for same part

Digestion of GFP and Double Terminator

Parts Information

Description BioBrick No. Well No. Length Plasmid
GFP BBa_E0040 1-14K 720bp pSB1A3
double terminator BBa_B0015 1-23L 129bp pSB1AK3
pSB1A3 pSB1A3 2157bp pSB1A3


Parts in wells 1-14K and pSB1A3 were purified with mycrocon. Part 1-23L was extracted from a gel previously.


  • Performed electrophoresis of 1-14K and 1-23L to estimate concentration of each solution.
  • Estimated concentration from photo of electrophoresis
  • pSB1A3 solution was done by other person.
  • Made digestion recipes(below) based on estimated concentrations
  • Made 30 ul of pSB1A3 solution, but latter found it insufficient to ligate parts
    • made more 50ul of it after


Part Well No. Amount
1-14K 200 ng/ul
1-23L 120 ng/ul
pSB1A3 2.5 ng/ul

Digestion

Reagent Amount
1-23L 0.5 uL
10x M buffer 5 uL
0.1%BSA 5 uL
Xba I 4 uL
Pst I 0.5 uL
DW 35 uL
Total 50 uL
Reagent Amount
1-14K 1.5 uL
10x H buffer 2 uL
0.1% BSA 2 uL
EcoR I 1 uL
Spe I 0.5 uL
DW 13 uL
Total 20 uL
Reagent Amount
pSB1A3 20 uL
10x H buffer 3 uL
0.1% BSA 3 uL
EcoR I 0.5 uL
Pst I 0.5 uL
DW 3 uL
Total 30 uL
Reagent Amount
pSB1A3 30 uL
10x H buffer 5 uL
0.1% BSA 5 uL
EcoR I 0.5 uL
Pst I 0.5 uL
DW 9 uL
Total 30 uL


  • Incubated each solution at 37C
  • Solution of 1-23L was incubated for 150 min
  • Solution of 1-14K was incubated for 90 min
  • 30 ul of pSB1A3 solution was incubated for 60 min
  • 50 ul of pSB1A3 solution was incubated for 30 min
  • Performed electrophoresis for each solution
  • put 12 uls each into wells of a gel like below.
Lane DNA
1 λ/HindIII,　EcoR I
2~3 1-14K
4~8 1-23L
9~16 pSB1A3

Results

  • Could not see bands of 1-23L because leaked out
    • Drove current for too long
  • Extracted the other samples from a gel using promega kit
  • Stored all at -20C.
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