Team:Warsaw/Calendar-Main/8 July 2009

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Gradient PCR Pho

Kama


Tasks:

  • Amplification of phoP/phoQ

Methods:

  • PCR mixture's composition:

    1ul pfu buffer (Fermentas), 1ul MgSO4 (Fermentas), 0,5ul primers, 0,5ul dNTPs (10 mM), 0,25ul pfu turbo polymerase, 0,5ul template DNA from Listeria, optionally: 0,75ul DMSO, solution was topped up with H2O to 10ul.
  • PCR programs:

    • pho

    4min 95°C 
     (30s 95°C, 1min 45-55°C, 4min 72°C)x3 
     (30s 95°C, 1min 55-60°C, 4min 72°C)x28 
     10min 72°C 
     ~ 7°C
  • Electrophoretic separation on 1% agarose gel

Results:

  • Gel (from left)
  1. control -
  2. 2-6 - samples (annealing temperature increases to from the left to the right)
  3. 7-11 - samples with DMSO (annealing temperature increases to from the left to the right)
  4. M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)

    5. Notes:


    Digest of plasmids pLG612-1 and pVDL9 (containing components of hemolisin secretion system)



    Kuba



    • pKS-CRO cut with XbaI and EcoRI

    • Gel (from the left)
    1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
    2. uncut plasmid (isolate no.1)
    3. cut plasmid (isolate no.1)
    4. uncut plasmid (isolate no.2)
    5. cut plasmid (isolate no.2)
    6. uncut plasmid (isolate no.3)
    7. cut plasmid (isolate no.3)
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