Biomod/2011/TUM/TNT/LabbookA/2011/09/01

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Real time PCR with MH255_647 and MH256_550 adding ErBr and Spermine

  • MH255_647 and MH256_550 are oligonucleotides of 18 nucleotides each, labeled with Atto fluorescent dyes Atto 647 and Atto 550.
  • The DNA intercalator ethidium bromide and the major groove binder Spermine are added at various concentrations accoring to their KD-Values.
  • The KD value of Spermin is 4,8µL and that of ethidium bromide is 12µL.
  • The concentrations of Spermin and ethidium bromide are as follows:
    • 1/19*KD, 1/9*KD, 1/4*KD, 1/2*KD, 1*KD, 2*KD, 4*KD, 9*KD, 19*KD.
  • Final concentration DNA = 10nM
  • Total volume = 100µL (filled up with 0,5 TBE buffer, 11mM MgCl2
  • Controls:
    • DNA MH255 and MH256 without fluorescent labels is used instead of MH255_647 and MH256_550 to check flourescence of spermin and ethidium bromide when interacting with DNA
    • Donor only: the strand MH255 without label and MH256_550 labeled with fluorescence donor are used
    • Acceptor only: the strand MH256 without label and MH255_550 labeled with fluorescence donor are used

Pipeting scheme:



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