Team:HokkaidoU Japan/Notebook/September16

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  • digestion of GFP(1-14K) and double terminator(1-23L)


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.


Method

  1. electrophoresed 1μl of 1-14K and 1-23L added 0.5μl of 6×sample buffer to estimate concentration of each solution.
  2. 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.
  3. made digestion recipes based on each concentrations.Why pSB1A3 recipe is two,because I firstly made 30μl of pSB1A3 solution,but I found it was insufficient to ligate parts,so I made more 50μl of it after.

Estimates of concentration

1-14K:200ng/μl

1-23L:120ng/μl

pSB1A3:2.5ng/μl


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

1.5μl

H-buffer2 μl

0.1%BSA2 μl

Eco1 μl

Spe0.5μl

DW13μl


pSB1A320μl

H-buffer3μl

0.1%BSA3 μl

Eco0.5 μl

Pst0.5μl

DW3μl


pSB1A330μl

H-buffer5μl

0.1%BSA5μl

Eco0.5 μl

Pst0.5μl

DW9μl


4)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,30μl of pSB1A3 solution was done about an hour,and 50μl of pSB1A3 was done about a half hour.


5)electrophoresed each solutions added 6×sample buffer.put 12μls each into wells of a gel.

1:λ/HindⅢ EcoRⅠ

2~3:1-14K

4~8:1-23L

9~16:pSB1A3


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