Team:Warsaw/Calendar-Main/27 August 2009

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Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil


Tasks:

  • Amplification of the mgtc promoter using new primers
  • Isolation of the pSB1A3 plasmid

Methods:

  • The PCR mix was prepared as follows:

    5μl buffer B (EURx)
    5μl 10mM dNTPs
    5μl forward starter
    5μl reverse starter
    2μl OptiTaq polymerase (EURx)
    2μl Salmonella matrix
    26 μl H2O
  • PCR programme:

     4min 95°C 
    (30s 95°C, 35s 56°C, 40s 72°C)x28
    10min 72°C
    ~ 4°C
  • The PCR results were visualised with gel electrophoresis on 1% agarose gel.
  • The plasmid isolation was carried out from 3μl of culture cultivated for 6h in 37°C. The Plasmid Mini kit (A&A Biotechnology) was used.

Results:

  • Nothing

Conclusions:

  • The temperature was chosen... poorly.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil


Tasks:

  • Creation of the cro-box double stranded DNA

Methods:

  • The mixture of equal volumes (10μl) of both ssDNA was heated to 94°C for 15min. and left to cool.

Assembly of endosomal detection operon

Marcin


Task 1:

Methods:

  • Reaction mixture composition:
  •  
    20 μl purified plasmid DNA product
    1 μl XbaI (Fermentas)
    1 μl PstI (Fermentas) in the case of C0051
    0.7 μl SpeI (Fermentas) in the case of E0022
    2 μl Buffer Tango (Fermentas)
    15 μl MQ water
  • Digest was performed about seven hours
  • After digestion samples were frozen

Making of the plac-RBS-llo-intA part

Jarek


Tasks:

  • Separation of digested DNA fragments in 0,8% agarose.

Results: