Team:HokkaidoU Japan/Notebook/September30

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*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3
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<u>parts information</u>
<u>parts information</u>
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Arabinose Promoter  1210bp(+primer:1259bp)  
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Arabinose Promoter  1210 bp(+primer:1259 bp)  
3-20B pSB2K3
3-20B pSB2K3
Line 15: Line 15:
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RBS+T3SSsignal  583bp(+primer:636bp)
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RBS+T3SSsignal  583 bp(+primer:636 bp)
PCRed in 29 September
PCRed in 29 September
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pSB1T3  2463bp(+primer:2504bp)
+
pSB1T3  2463 bp(+primer:2504 bp)
PCRed in 26 August
PCRed in 26 August
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== Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3 ==
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= Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3 =
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==digestion mix==
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1.digestion mix was made according to the table below
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{|style="text-align:center; float:left;" class="protocol"
{|style="text-align:center; float:left;" class="protocol"
!Reagent
!Reagent
Line 102: Line 101:
|style="border-top:1px solid #996;"|'''50 uL'''
|style="border-top:1px solid #996;"|'''50 uL'''
|}
|}
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2.incubated at 37C for an hour
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<div style="clear:both"></div>
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3.electrophoresed 3 samples with 6uL λ/HindⅢ EcoRI
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写真:9/30　14:01
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4.extracted 3 samples from a gel
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5.added 4.5 ul 3M CH3COONa
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+
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6.added 125 uL 100% EtOH
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+
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7.centrifuged at 4C,15000rpm for 5min
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8.discarded the supertenant
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9.added 100 uL 70% EtOH
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10.centrifuged at 4C,15000rpm for 5min
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11.dry up the samples
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+
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12.dissolved the samples with 2 uL TE
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13.mixed the 3 samples
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14.added 6 uL Mighty Mix
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15.incubated at 16C for 30 min
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16.put the sample into 100 uL competent cell
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+
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17.incubated at 0C for 30 min
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+
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18.heatshocked at 42C for 60 sec
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19.incubated at 0C for 5min
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20.added 400 uL SOB
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21.incubated at 30C for an hour and a half
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22.incubated at 37C for 45min
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23.
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# incubated at 37C for 1h
 +
# electrophoresed 3 samples with 6 uL λ/''Hin''dIII EcoR I
 +
# extracted 3 samples from a gel
 +
# added 4.5 uL of 3 M CH<sub>3</sub>COONa
 +
# added 125 uL 100% EtOH
 +
# centrifuged at 4C,15000 rpm for 5 min
 +
# discarded the supertenant
 +
# added 100 uL 70% EtOH
 +
# centrifuged at 4C,15000 rpm for 5 min
 +
# dried up the samples
 +
# dissolved the samples with 2 uL of TE
 +
# mixed the 3 samples
 +
# added 6 uL Mighty Mix (Ligation Kit)
 +
# incubated at 16C for 30 min
 +
# put the sample into 100 uL competent cell
 +
# incubated at 0C for 30 min
 +
# heatshocked at 42C for 60 sec
 +
# incubated at 0C for 5 min
 +
# added 400 uL of SOB
 +
# incubated at 30C for 1.5 hrs
 +
# incubated at 37C for 45 min
 +
# plated the sample on LBT medium

Latest revision as of 13:00, 27 October 2010

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


parts information

Arabinose Promoter 1210 bp(+primer:1259 bp)

3-20B pSB2K3

PCRed in 21 September


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

PCRed in 29 September

pSB1T3 2463 bp(+primer:2504 bp)

PCRed in 26 August


Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3

digestion mix

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
  1. incubated at 37C for 1h
  2. electrophoresed 3 samples with 6 uL λ/HindIII EcoR I
  3. extracted 3 samples from a gel
  4. added 4.5 uL of 3 M CH3COONa
  5. added 125 uL 100% EtOH
  6. centrifuged at 4C,15000 rpm for 5 min
  7. discarded the supertenant
  8. added 100 uL 70% EtOH
  9. centrifuged at 4C,15000 rpm for 5 min
  10. dried up the samples
  11. dissolved the samples with 2 uL of TE
  12. mixed the 3 samples
  13. added 6 uL Mighty Mix (Ligation Kit)
  14. incubated at 16C for 30 min
  15. put the sample into 100 uL competent cell
  16. incubated at 0C for 30 min
  17. heatshocked at 42C for 60 sec
  18. incubated at 0C for 5 min
  19. added 400 uL of SOB
  20. incubated at 30C for 1.5 hrs
  21. incubated at 37C for 45 min
  22. plated the sample on LBT medium
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