Template:Team:KULeuven/18 August 2009/VanillinProduction

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* Re-plated the ligation products (SAMS and EF)
* Re-plated the ligation products (SAMS and EF)
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* We propose 3 tests to check if the restriction digest (esp. between EcoRI and XbaI, because they lie very close to each other) works properly:
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* We propose 3 tests to check if the restriction digest (esp. between EcoRI and XbaI, because they lie very close together) works properly:
-
** Cut every plasmid (Sam8, Sam5, Ech, Fcs) separately with EcoRI, XbaI and -only Sam8 and Fcs- with SpeI.
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'''Test 1''': Cut every plasmid (''sam8, sam5, ech, Fcs'') separately with EcoRI, XbaI and -only ''sam8'' and ''fcs''- with SpeI.
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** Cut subsequently with EcoRI and XbaI, leaving an incubation period of 1h between two digests. Total: 30µl
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** Use pUC18 vector
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'''Test 2''': Cut subsequently with EcoRI and XbaI, leaving an incubation period of 1h between two digests. Total volume: 30µl, to dilute glycerol (which inhibits restriction)
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* Made fluid cultures of Sam8, Sam5, Ech, Fcs
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'''Test 3''': Use pUC18 vector
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* Made fluid cultures of ''sam8, sam5, ech, fcs''

Latest revision as of 11:23, 14 September 2009

  • Cells didn't grow. Yet again...
  • Re-plated the ligation products (SAMS and EF)
  • We propose 3 tests to check if the restriction digest (esp. between EcoRI and XbaI, because they lie very close together) works properly:

Test 1: Cut every plasmid (sam8, sam5, ech, Fcs) separately with EcoRI, XbaI and -only sam8 and fcs- with SpeI.

Test 2: Cut subsequently with EcoRI and XbaI, leaving an incubation period of 1h between two digests. Total volume: 30µl, to dilute glycerol (which inhibits restriction)

Test 3: Use pUC18 vector

  • Made fluid cultures of sam8, sam5, ech, fcs
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