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Real Time PCR with MH_255_647N and MH_256_550 adding Ethidium Bromide and Spermine


  • The DNA intercalator ethidium bromide (EtBr) and the major groove binder spermine are added in variant concentrations according to their KD values (see references).
  • The KD value of spermin is 4,8 µM and that of ethidium bromide is 12 µM.
  • 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 DNA concentration = 10 nM
  • Total volume = 100 µL (filled up with 0,5 TBE buffer, 11mM MgCl2)
  • Controls:
    • DNA MH_255 and MH_256 without fluorescent labels is used instead of MH_255_647N and MH_256_550 to check flourescence of spermin and ethidium bromide when interacting with DNA
    • Donor only: the strand MH_255 without label and MH_256_550 labeled with fluorescence donor are used
    • Acceptor only: the strand MH_256 without label and MH_255_647N labeled with fluorescence donor are used
  • Concentrations of stock solutions:
    • Spermin: 50 µM and 200 µM
    • EtBr: 50 µM and 200 µM
  • Concentrations of DNA solutions:
    • MH_255_647N: 2,8 µM
    • MH_256_550: 7,1 µM
  • dilution 1:10 in buffer is used for real time PCR
    • MH_255 (without label for control): 100 µM
    • MH_256 (without label for control): 100 µM

-> dilution 1:100 in buffer is used for real time PCR

Table 1: Pipeting scheme spermine

Concentration Spermine Spermine Stock [µL] MH255_647 0,28 µM [µL] MH256_550 0,71 µM [µL] Buffer (0,5 TBE, 11 mM MgCl2)
1/19*KD 0,51 (50 µM stock) 3,6 1,4 94,49
1/9*KD 1,07 (50 µM stock) 3,6 1,4 93,93
1/4*KD 2,4 (50 µM stock) 3,6 1,4 92,6
1/2*KD 4,8 (50 µM stock) 3,6 1,4 90,2
1*KD 9,6 (50µM stock) 3,6 1,4 85,4
2*KD 19,2 (50 µM stock) 3,6 1,4 75,8
4*KD 9,6 (200 µM stock) 3,6 1,4 85,4
9*KD 21,6 (200 µM stock) 3,6 1,4 73,4
19*KD 45,6 (200 µM stock) 3,6 1,4 49,4

Table 2: Pipeting scheme ethidium bromide.

Concentration EtBr EtBr Stock [µL] MH255_647 0,28 µM [µL] MH256_550 0,71 µM [µL] Buffer (0,5 TBE, 11 mM MgCl2)
0,021*KD 0,51 (50 µM stock) 3,6 1,4 94,49
0,044*KD 1,07 (50 µM stock) 3,6 1,4 93,93
0,1*KD 2,4 (50 µM stock) 3,6 1,4 92,6
0,2*KD 4,8 (50 µM stock) 3,6 1,4 90,2
0,4*KD 9,6 (50 µM stock) 3,6 1,4 85,4
0,8*KD 19,2 (50 µM stock) 3,6 1,4 75,8
1,6*KD 9,6 (200 µM stock) 3,6 1,4 85,4
3,6*KD 21,6 (200 µM stock) 3,6 1,4 73,4
7,6*KD 45,6 (200 µM stock) 3,6 1,4 49,4

Table 3: Pipeting scheme ethidium bromide control without labels: Error: there was added 1 µL of buffer too much.

Concentration EtBr EtBr Stock [µL] MH_255 1 µM [µL] MH_256 1 µM [µL] Buffer (0,5 TBE, 11 mM MgCl2)
0,021*KD 0,51 (50 µM stock) 1 1 98,49 (=1 µL too much!)
0,044*KD 1,07 (50 µM stock) 1 1 97,93
0,1*KD 2,4 (50 µM stock) 1 1 96,6
0,2*KD 4,8 (50 µM stock) 1 1 94,2
0,4*KD 9,6 (50 µM stock) 1 1 89,4
0,8*KD 19,2 (50 µM stock) 1 1 79,8
1,6*KD 9,6 (200 µM stock) 1 1 89,4
3,6*KD 21,6 (200 µM stock) 1 1 77,4
7,6*KD 45,6 (200 µM stock) 1 1 53,4
  • For hybridisation of DNA helix, the samples are incubated at 65 °C. The temperature is lowered by 1 °C every minute. Fluorescence is meausured at 25 °C every two minutes for two hours.

Atto 550 was excited at 545 nm and fluorescence detected at 568 nm, Atto 647N was excited at 635 nm and detected 665 nm, thus matching the conditions of the real time PCR's optical filters.

Results

The following graphs show no significant correlation between neither spermine nor ethidium bromide concentration and FRET efficiency in this experiment. A concentration of labeled oligos of 10 nM seems to be below the detection limit of the real time PCR gadget. Additionally, it should be noted that the filters do not excite the fluorescent dyes and measure fluorescence at wavelenghts that are optimal for Atto 550 and Atto 647N (see above).

FRET efficiency Spermine skal.PNGFRET efficiency EtBr.PNG