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:
    • Spermin: 1/19*KD, 1/9*KD, 1/4*KD, 1/2*KD, 1*KD, 2*KD, 4*KD, 9*KD, 19*KD.
    • EtBr: 0,021*KD, 0,044*KD, 0,1*KD, 0,2*KD, 0,4*KD, 0,8*KD, 1,6*KD, 3,6*KD, 7,6*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
  • Concentrations of stock solutions:
    • Spermin: 50µM and 200µM
    • EtBr: 50µM and 200µM

Pipeting scheme spermine:

concentration spermine Spermin Stock [µL] MH255_647[µL] MH256_550[µL] buffer (0,5 TBE, 11mM MgCl2)
1/19*KD 0,51 (50µM stock) 3,6 1,4 94,49
1/9*KD 1,07 (50µM stock) 3,6 1,4 93,93
1/4*KD 2,4 (50µM stock) 3,6 1,4 92,6
1/2*KD 4,8 (50µM stock) 3,6 1,4 90,2
1*KD 9,6 (50µM stock) 3,6 1,4 85,4
2*KD 19,2 (50µM stock) 3,6 1,4 75,8
4*KD 9,6 (200µM stock) 3,6 1,4 85,4
9*KD 21,6 (200µM stock) 3,6 1,4 73,4
19*KD 45,6 (200µM stock) 3,6 1,4 49,4

Pipeting scheme spermine control without labels: Error: there was added 1 µL of buffer too much

concentration spermine Spermin Stock [µL] MH255[µL] MH256[µL] buffer (0,5 TBE, 11mM MgCl2)
1/19*KD 0,51 (50µM stock) 1 1 98,49 (=1µL too much!)
1/9*KD 1,07 (50µM stock) 1 1 97,93
1/4*KD 2,4 (50µM stock) 1 1 96,6
1/2*KD 4,8 (50µM stock) 1 1 94,2
1*KD 9,6 (50µM stock) 1 1 89,4
2*KD 19,2 (50µM stock) 1 1 79,8
4*KD 9,6 (200µM stock) 1 1 89,4
9*KD 21,6 (200µM stock) 1 1 77,4
19*KD 45,6 (200µM stock) 1 1 53,4


Pipeting scheme Ethidium Bromide:

concentration EtBr EtBr Stock [µL] MH255_647[µL] MH256_550[µL] buffer (0,5 TBE, 11mM MgCl2)
0,021*KD 0,51 (50µM stock) 3,6 1,4 94,49
0,044*KD 1,07 (50µM stock) 3,6 1,4 93,93
0,1*KD 2,4 (50µM stock) 3,6 1,4 92,6
0,2*KD 4,8 (50µM stock) 3,6 1,4 90,2
0,4*KD 9,6 (50µM stock) 3,6 1,4 85,4
0,8*KD 19,2 (50µM stock) 3,6 1,4 75,8
1,6*KD 9,6 (200µM stock) 3,6 1,4 85,4
3,6*KD 21,6 (200µM stock) 3,6 1,4 73,4
7,6*KD 45,6 (200µM stock) 3,6 1,4 49,4

Pipeting scheme ethidium bromide control without labels: Error: there was added 1 µL of buffer too much

concentration EtBr EtBr Stock [µL] MH255[µL] MH256[µL] buffer (0,5 TBE, 11mM MgCl2)
0,021*KD 0,51 (50µM stock) 1 1 98,49 (=1µL too much!)
0,044*KD 1,07 (50µM stock) 1 1 97,93
0,1*KD 2,4 (50µM stock) 1 1 96,6
0,2*KD 4,8 (50µM stock) 1 1 94,2
0,4*KD 9,6 (50µM stock) 1 1 89,4
0,8*KD 19,2 (50µM stock) 1 1 79,8
1,6*KD 9,6 (200µM stock) 1 1 89,4
3,6*KD 21,6 (200µM stock) 1 1 77,4
7,6*KD 45,6 (200µM stock) 1 1 53,4


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