Team:HokkaidoU Japan/Notebook/September16

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Notebook

Digestion of GFP and Double Terminator

Parts Information

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 37℃ 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 6×sample buffer.put 12uls each into wells of a gel.
1 λ/HindIII,　EcoR I
2~3 1-14K
4~8 1-23L
9~16 pSB1A3


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