NYMU Taipei/Lab Notes/2007 9 1

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*Plasmid extraction: RBS+cinR 1, RBS+HSL 1, RBS+HSL 2, RBS+HSL 3 from 5ml LB culture to 50ul EB
*Plasmid extraction: RBS+cinR 1, RBS+HSL 1, RBS+HSL 2, RBS+HSL 3 from 5ml LB culture to 50ul EB
*The concentration was measured with spectrophotometer at 260nm
*The concentration was measured with spectrophotometer at 260nm
-
**RBS+cinR 1: 8ug/ul
+
**RBS+cinR 1: 0.08ug/ul
-
**RBS+HSL 1: 3ug/ul
+
**RBS+HSL 1: 0.03ug/ul
-
**RBS+HSL 2: 8ug/ul
+
**RBS+HSL 2: 0.08ug/ul
-
**RBS+HSL 3: 6ug/ul
+
**RBS+HSL 3: 0.06ug/ul
*Enzyme digest RBS+cinR 1
*Enzyme digest RBS+cinR 1
**RBS+cinR 1: 14ul
**RBS+cinR 1: 14ul
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**There seems to be some incomplete digestion of RBS+HSL 2, possibly result from too many DNA.
**There seems to be some incomplete digestion of RBS+HSL 2, possibly result from too many DNA.
**BsaBI digestion test of RBS+cinR failed. The lower bright band is probably negative supercoil form of the plasmid. Redo ligation RBS+cinR ver. 2.
**BsaBI digestion test of RBS+cinR failed. The lower bright band is probably negative supercoil form of the plasmid. Redo ligation RBS+cinR ver. 2.
 +
**Assuming that the restriction enzyme is working, it should digest 1ug of DNA in 5min according to the NEB catalog.
**Todo: BsaBI digestion of cinR
**Todo: BsaBI digestion of cinR
*Ligation (RBS+cinR+RBS+HSL)
*Ligation (RBS+cinR+RBS+HSL)
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**10x ligase (Yeastern, 2U/ul): 1ul
**10x ligase (Yeastern, 2U/ul): 1ul
**H2O: 4ul
**H2O: 4ul
-
**RT 20min
+
**Incubated at RT for 20min
*Ligation (RBS+cinR ver. 2)
*Ligation (RBS+cinR ver. 2)
**RBS: 1ul
**RBS: 1ul
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**10x ligase (Yeastern, 2U/ul): 1ul
**10x ligase (Yeastern, 2U/ul): 1ul
**H2O: 6ul
**H2O: 6ul
-
**RT 20min
+
**Incubated at RT for 20min

Latest revision as of 06:23, 16 September 2007

  • Plasmid extraction: RBS+cinR 1, RBS+HSL 1, RBS+HSL 2, RBS+HSL 3 from 5ml LB culture to 50ul EB
  • The concentration was measured with spectrophotometer at 260nm
    • RBS+cinR 1: 0.08ug/ul
    • RBS+HSL 1: 0.03ug/ul
    • RBS+HSL 2: 0.08ug/ul
    • RBS+HSL 3: 0.06ug/ul
  • Enzyme digest RBS+cinR 1
    • RBS+cinR 1: 14ul
    • SpeI: 1ul
    • PstI 1ul
    • 10x buffer 2: 2ul
    • 10x BSA: 2ul
    • 37°C water bath, 2h
    • Heat inactivation: 65°C, 20min
  • Enzyme digest RBS+HSL 2
    • RBS+HSL 2: 14ul
    • XbaI: 1ul
    • PstI: 1ul
    • 10x buffer 3: 2ul
    • 10x BSA: 2ul
    • 37°C water bath, 2h
    • Heat inactivation: 65°C, 20min
  • Enzyme digest RBS+HSL 3
    • RBS+HSL 3: 14ul
    • XbaI: 1ul
    • PstI: 1ul
    • 10x buffer 3: 2ul
    • 10x BSA: 2ul
    • 37°C water bath, 2h
    • Heat inactivation: 65°C, 20min
  • Check RBS+cinR 1 with BsaBI digestion
    • RBS+cinR 1: 3ul
    • BsaBI: 1ul
    • 10x buffer 2: 2ul
    • H2O: 14ul
    • 60°C dry bath, 1h

NYMU Taipei Gel 20070901 plasmid check 1.JPG

  • Well 1: 100pb ladder
  • Well 2 3: "RBS+HSL 2" XbaI, PstI
  • Well 4 5: "RBS+HSL 3" XbaI, PstI
  • Well 6 7: "RBS+cinR 1" SpeI, PstI
  • Well 8: "RBS+cinR 1" BsaBI
  • Comment
    • There seems to be some incomplete digestion of RBS+HSL 2, possibly result from too many DNA.
    • BsaBI digestion test of RBS+cinR failed. The lower bright band is probably negative supercoil form of the plasmid. Redo ligation RBS+cinR ver. 2.
    • Assuming that the restriction enzyme is working, it should digest 1ug of DNA in 5min according to the NEB catalog.
    • Todo: BsaBI digestion of cinR
  • Ligation (RBS+cinR+RBS+HSL)
    • RBS+cinR: 1ul
    • RBS+HSL 3: 3ul
    • 10x buffer A: 1ul
    • 10x ligase (Yeastern, 2U/ul): 1ul
    • H2O: 4ul
    • Incubated at RT for 20min
  • Ligation (RBS+cinR ver. 2)
    • RBS: 1ul
    • cinR: 1ul
    • 10x buffer A: 1ul
    • 10x ligase (Yeastern, 2U/ul): 1ul
    • H2O: 6ul
    • Incubated at RT for 20min