Team:HokkaidoU Japan/Notebook/September17

From 2010.igem.org

(Difference between revisions)
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|-
|-
<!-- Row 1 -->
<!-- Row 1 -->
-
<!--      Description -->|
+
<!--      Description -->|GFP
-
<!--      BioBrick No. -->|
+
<!--      BioBrick No. -->|BBa_E0040
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<!--          Well No. -->|
+
<!--          Well No. -->|1-14K
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<!--            Length -->|
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<!--            Length -->|720bp
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<!--          Plasmid -->|
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<!--          Plasmid -->|pSB1A3
|-
|-
<!-- Row 3 -->
<!-- Row 3 -->
-
<!--      Description -->|
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<!--      Description -->|double terminator
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<!--      BioBrick No. -->|
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<!--      BioBrick No. -->|BBa_B0015
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<!--          Well No. -->|
+
<!--          Well No. -->|1-23L
-
<!--            Length -->|
+
<!--            Length -->|129bp
-
<!--          Plasmid -->|
+
<!--          Plasmid -->|pSB1AK3
|-
|-
<!-- Row 3 -->
<!-- Row 3 -->
-
<!--      Description -->|
+
<!--      Description -->|pSB1A3
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<!--      BioBrick No. -->|
+
<!--      BioBrick No. -->|pSB1A3
<!--          Well No. -->|
<!--          Well No. -->|
-
<!--            Length -->|
+
<!--            Length -->|2157bp
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<!--          Plasmid -->|
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<!--          Plasmid -->|pSB1A3
|-
|-
|}
|}
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{| class="protocol"
 
-
|-
 
-
|GFP
 
-
|BBa_E0040
 
-
|1-14K
 
-
|720bp
 
-
|pSB1A3
 
-
|-
 
-
|double terminator
 
-
|BBa_B0015
 
-
|1-23L
 
-
|129bp
 
-
|pSB1AK3
 
-
|-
 
-
|pSB1A3
 
-
|pSB1A3
 
-
| -
 
-
|2157bp
 
-
|pSB1A3
 
-
|}
 
-
1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.
+
Parts in wells 1-14K and pSB1A3 were purified with mycrocon. Part 1-23L was extracted from a gel previously.
-
* electrophoresed 1ul of 1-14K and 1-23L added 0.5ul of 6×sample buffer to estimate concentration of each solution.
+
* Performed electrophoresis of 1-14K and 1-23L to estimate concentration of each solution.
-
* estimated concentration from photo of electrophoresys. But I forgot to electrophorese pSB1A3 solution with the other samples, so pSB1A3 solution was done by other person.
+
* Estimated concentration from photo of electrophoresis
-
* made digestion recipes based on each concentrations(below). Why pSB1A3 recipe is two, because I firstly made 30ul of pSB1A3 solution, but I found it was insufficient to ligate parts, so I made more 50ul of it after.
+
* pSB1A3 solution was done by other person.
 +
* Made digestion recipes(below) based on estimated concentrations
 +
* Made 30ul of pSB1A3 solution, but latter found it insufficient to ligate parts
 +
** made more 50ul of it after
 +
 
{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|1-14K
|1-14K
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===Digestion Menu===
===Digestion Menu===
{|style="text-align:center; float:left;" class="protocol"
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|1-23L
|1-23L
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{|style="text-align:center; float:left;" class="protocol"
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|1-14K
|1-14K
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{|style="text-align:center; float:left;" class="protocol"
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|pSB1A3
|pSB1A3
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{|style="text-align:center; float:left;" class="protocol"
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|pSB1A3
|pSB1A3
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<div style="clear:both"></div>
<div style="clear:both"></div>
-
* put each solutions into 37C incubator.
+
 
-
* 1-23L solution was put about a half and two hours, 1-14K solution was done about a half and an hour, 30ul of pSB1A3 solution was done about an hour, and 50ul of pSB1A3 was done about a half hour.
+
 
-
* electrophoresed each solutions added 6x sample buffer.
+
* Incubated each solution at 37C
 +
* Solution of 1-23L was incubated for 150 min
 +
* Solution of 1-14K was incubated for 90 min
 +
* 30ul of pSB1A3 solution was incubated for 60 min
 +
* 50ul of pSB1A3 solution was incubated for 30 min
 +
* Performed electrophoresis for each solution
 +
 
* put 12uls each into wells of a gel like below.
* put 12uls each into wells of a gel like below.
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|-
|-
|1
|1
-
|λ/''Hin''dIII,　EcoR I
+
|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII,　EcoR I]
|-
|-
|2~3
|2~3
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'''Result'''
'''Result'''
-
* cannot see 1-23L because of overflowing.
+
* Could not see bands of 1-23L because leaked out
-
* extracted the other samples from a gel.
+
** Drove current for too long
-
* dissolve them with 50 ul of Nuclease free water, and they were stocked to freeze in -20C.
+
* Extracted the other samples from a gel using promega kit
 +
* Stored at -20C.

Revision as of 18:07, 19 September 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 30ul of pSB1A3 solution, but latter found it insufficient to ligate parts
    • made more 50ul of it after


Reagent Amount
1-14K 200 ng/ul
1-23L 120 ng/ul
pSB1A3 2.5 ng/ul

Digestion Menu

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

Result

  • 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 at -20C.
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