Team:Warsaw/Calendar-Main/16 July 2009

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Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil


Tasks:

  • Transformant selection

Methods:

  • White colonies were picked up and transferred to a new petri dish.
  • Liquid cultures were established along the way for plasmid purification.
  • Both the dish and the liquid cultures were incubated at 37°C overnight.

Results:

  • All of the transferred colonies turned blue overnight and the purified plasmids yealded no mgtc promoter.

Cloning of p53 coding sequence

Marcin

Task:

  • Transformation of chemocompetent E. coli strain DH5α

Methods:

  • Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
  • Detailed protocol of transformation is described here. The only modification is usage of half volume of ligation mixture prepared 15.07.09

Assembly of endosomal detection operon

Marcin

Task:

  • Restriction digest of biobricks

Comment:

Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:

BBa_B0032

BBa_c0040

BBa_C0051

BBa_E0032


Methods:

  • Digest of BBa_B0032 using SpeI and PstI
    • Reaction mixture composition:
      10 μl purified plasmid DNA product
      0.5 μl SpeI (Fermentas)
      1 μl PstI (Fermentas)
      5 μl Buffer Tango (Fermentas)
      34 μl MQ water
  • Digest of other biobricks using PstI and XbaI
    • Reaction mixture composition:
      10 μl purified plasmid DNA product
      1 μl XbaI (Fermentas)
      1 μl PstI (Fermentas)
      5 μl Buffer Tango (Fermentas)
      34 μl MQ water
  • Both reaction were performed overnight (~12 hours) and both of them were subsequently inactivated via heating in 80°C for 20 minutes

Construction of K177012 operon1_part2

Ania

Tasks:

  • Alkaline lysis of bacterial cultures to obtain plasmid.


Testing different E. coli strains regarding lacI and AraC repressors

Franek


Tasks:


Methods:

  • 2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing BBa_K177024 and BBa_K177025 on pSB1A2 plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.
  • Chemocompetent E. coli cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains (prepered earlier by Kuba) were transformed according to our standard procedure with either BBa_K177024 or BBa_K177025


Results:

  • Will be determined by cell colonies presence on plates

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