Team:HokkaidoU Japan/Notebook/September30

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Notebook
  • Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
  • Transformation


parts information

Arabinose Promoter 1210bp(+primer:1259bp)

3-20B pSB2K3

PCRed in 21 September


RBS+T3SSsignal 583bp(+primer:636bp)

PCRed in 29 September

pSB1T3 2463bp(+primer:2504bp)

PCRed in 26 August


Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3

1.digestion mix was made according to the table below

Reagent Amount
10x H buffer 2 uL
DW 1.6
pSB1T3 1
BSA 2
EcoR I 0.2
Pst I 0.2
Total 20 uL
Reagent Amount
10x H buffer 2 uL
DW 11.7
Promoter 3.5
BSA 2
EcoR I 0.5
Spe I 0.3
Total 20 uL
Reagent Amount
10x M buffer 5 uL
DW 32.8
T3SSsignal 2.5
BSA 5
Xba I 4.5
Pst I 0.2
Total 50 uL

2.incubated at 37C for an hour

3.electrophoresed 3 samples with 6uL λ/HindⅢ EcoRI

写真:9/30　14:01


4.extracted 3 samples from a gel

5.added 4.5 ul 3M CH3COONa

6.added 125 uL 100% EtOH

7.centrifuged at 4C,15000rpm for 5min

8.discarded the supertenant

9.added 100 uL 70% EtOH

10.centrifuged at 4C,15000rpm for 5min

11.dry up the samples

12.dissolved the samples with 2 uL TE

13.mixed the 3 samples

14.added 6 uL Mighty Mix

15.incubated at 16C for 30 min

16.put the sample into 100 uL competent cell

17.incubated at 0C for 30 min

18.heatshocked at 42C for 60 sec

19.incubated at 0C for 5min

20.added 400 uL SOB

21.incubated at 30C for an hour and a half

22.incubated at 37C for 45min

23.
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