Team:HokkaidoU Japan/Notebook/September17

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*Construction of GFP marker for a part which will be secreted using T3SS
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*Ordered primers for construction for same part
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== Digestion of GFP and Double Terminator ==
== Digestion of GFP and Double Terminator ==
===Parts Information===
===Parts Information===
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{| class="protocol"
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|-
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!Description
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!BioBrick No.
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!Well No.
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!Length
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!Plasmid
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|-
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<!--          Well No. -->|
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<!--            Length -->|
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|-
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|-
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{| class="protocol"
{| class="protocol"
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Revision as of 17:44, 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

1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.


  • electrophoresed 1ul of 1-14K and 1-23L added 0.5ul of 6×sample buffer 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.
  • 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.
1-14K 200 ng/ul
1-23L 120 ng/ul
pSB1A3 2.5 ng/ul

Digestion Menu

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
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
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
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
  • 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.
  • 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

  • cannot see 1-23L because of overflowing.
  • extracted the other samples from a gel.
  • dissolve them with 50 ul of Nuclease free water, and they were stocked to freeze in -20C.
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