Team:HokkaidoU Japan/Notebook/September17

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 +
*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===
 +
{| class="protocol"
 +
|-
 +
!Description
 +
!BioBrick No.
 +
!Well No.
 +
!Length
 +
!Plasmid
 +
|-
 +
<!-- Row 1 -->
 +
<!--      Description -->|GFP
 +
<!--      BioBrick No. -->|BBa_E0040
 +
<!--          Well No. -->|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]]
 +
<!--            Length -->|720bp
 +
<!--          Plasmid -->|pSB1A3
 +
|-
 +
<!-- Row 3 -->
 +
<!--      Description -->|double terminator
 +
<!--      BioBrick No. -->|BBa_B0015
 +
<!--          Well No. -->|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
 +
<!--            Length -->|129bp
 +
<!--          Plasmid -->|pSB1AK3
 +
|-
 +
<!-- Row 3 -->
 +
<!--      Description -->|pSB1A3
 +
<!--      BioBrick No. -->|pSB1A3
 +
<!--          Well No. -->|
 +
<!--            Length -->|2157bp
 +
<!--          Plasmid -->|pSB1A3
 +
|-
 +
|}
 +
 
 +
 
 +
Parts in wells 1-14K and pSB1A3 were purified with mycrocon. Part 1-23L was extracted from a gel previously.
 +
 
 +
 
 +
* 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
 +
* 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
 +
 
 +
 
 +
{|style="text-align:center;" class="protocol"
 +
|-
 +
!Part Well No.
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-14K]]
 +
|200 ng/ul
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
 +
|120 ng/ul
 +
|-
 +
|pSB1A3
 +
|2.5 ng/ul
 +
|}
 +
 
 +
===Digestion===
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|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
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''50 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|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
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''20 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!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
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''30 uL'''
 +
|}
 +
 
 +
{|style="text-align:center; float:left;" class="protocol"
 +
|-
 +
!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
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''30 uL'''
 +
|}
 +
 
 +
<div style="clear:both"></div>
 +
 
 +
 
 +
 
 +
* Incubated each solution at 37C
 +
* Solution of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] was incubated for 150 min
 +
* Solution of [[Team:HokkaidoU_Japan/Parts#BioBricks|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.
 +
 
 +
{|class="protocol"
 +
|'''Lane'''
 +
|'''DNA'''
 +
|-
 +
|1
 +
|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII,　EcoR I]
 +
|-
 +
|2~3
 +
|1-14K
 +
|-
 +
|4~8
 +
|1-23L
 +
|-
 +
|9~16
 +
|pSB1A3
 +
|}
 +
 
 +
'''Results'''
 +
* Could not see bands of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]] because leaked out
 +
** Drove current for too long
 +
* Extracted the other samples from a gel using promega kit
 +
* 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.
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September17"