Team:Warsaw/Calendar-Main/21 April 2009

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Cloning of hly gene into pKSII+ vector

Kamil


Tasks:

  • Amplification of hly
  • Measurement of concentration of isolates from Yersinia and Listeria

Methods:

  • PCR mixture's composition:
    2ul pfu buffer (Fermentas) 
    1ul MgSO4 (Fermentas) 
    0,5ul primers 
    1,5ul dNTPs (10 mM, 
    01,ul pfu polymerase (Fermentas) 
    1ul template DNA from Listeria

Solution was topped up with H2O to 20ul.

  • PCR program:
  • hly

    300s 95°C 
    (30s 95°C, 35s 42°C, 150s 72°C)x2
    (30s 95°C, 35s 47°C, 150s 72°C)x28
    600s 72°C
    ~ 4°C
  • Electrophoretic separation on 1% agarose gel
  • Measurement of concentration of isolates on NanoDrop

Results:

  • Gel (from left)
  1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
  2. hly
  3. hly control -
  • Measurement from NanoDrop: + Yersinia - 29,6 ng/ul + Listeria - 50,7 ng/ul

Conclusions:

  • 200ng of DNA should be used on sample
  • Time of anealing should be prolonged to 45s
  • Concentration of Mg should be increased

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