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 according 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
- Concentrations of DNA solutions:
- MH255_647N: 2,8µM
- MH256_550: 7,1µM
-> dilution 1:10 in buffer is used for RtPCR
- MH255 (without label for control): 100µM
- MH256 (without label for control): 100µM
-> dilution 1:100 in buffer is used for RtPCR
Pipeting scheme spermine:
| concentration spermine
|
Spermin Stock [µL]
|
MH255_647 0,28µM [µL]
|
MH256_550 0,71µM [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 1µM [µL]
|
MH256 1µM [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 0,28µM [µL]
|
MH256_550 0,71µM [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 1µM [µL]
|
MH256 1µM [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
|
- 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 545nm and fluorescence detected at 568nm, Atto 647N was excited at 635nm and detected 665nm, thus matching the conditions of the RT-PCRs optical filters.
Results
The following links show that no significant connection between spermine or Ethidium Bromide concentration and FRET efficiency could be detected in this experiment. A concentration of labeled oligos of 10 nM seems to be below detection limit of the Real Time PCR machine. 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)
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Project name
