Biomod/2011/TUM/TNT/LabbookA/2011/09/01: Difference between revisions

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==Real time PCR with MH255_647 and MH256_550 adding ErBr and Spermine==
<center><h1>'''1<font size="-1">st</font> Sep 2011'''</h1></center>
* 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 K<sub>D</sub>-Values.
<h2> Real time PCR with [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#MH_255_and_MH_256 MH_255_647N and MH_256_550] adding ethidium bromide and spermine</h2>
* The K<sub>D</sub> value of Spermin is 4,8µM and that of ethidium bromide is 12µM.
 
* The concentrations of Spermin and ethidium bromide are as follows:
 
* The DNA intercalator ethidium bromide (EtBr) and the major groove binder spermine are added in variant concentrations according to their K<sub>D</sub> values.
* The K<sub>D</sub> 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*K<sub>D</sub>, 1/9*K<sub>D</sub>, 1/4*K<sub>D</sub>, 1/2*K<sub>D</sub>, 1*K<sub>D</sub>, 2*K<sub>D</sub>, 4*K<sub>D</sub>, 9*K<sub>D</sub>, 19*K<sub>D</sub>.
**Spermin: 1/19*K<sub>D</sub>, 1/9*K<sub>D</sub>, 1/4*K<sub>D</sub>, 1/2*K<sub>D</sub>, 1*K<sub>D</sub>, 2*K<sub>D</sub>, 4*K<sub>D</sub>, 9*K<sub>D</sub>, 19*K<sub>D</sub>.
**EtBr: 0,021*K<sub>D</sub>, 0,044*K<sub>D</sub>, 0,1*K<sub>D</sub>, 0,2*K<sub>D</sub>, 0,4*K<sub>D</sub>, 0,8*K<sub>D</sub>, 1,6*K<sub>D</sub>, 3,6*K<sub>D</sub>, 7,6*K<sub>D</sub>.
**EtBr: 0,021*K<sub>D</sub>, 0,044*K<sub>D</sub>, 0,1*K<sub>D</sub>, 0,2*K<sub>D</sub>, 0,4*K<sub>D</sub>, 0,8*K<sub>D</sub>, 1,6*K<sub>D</sub>, 3,6*K<sub>D</sub>, 7,6*K<sub>D</sub>.
* Final concentration DNA = 10nM
* Final DNA concentration = 10 nM
* Total volume = 100µL (filled up with 0,5 TBE buffer, 11mM MgCl<sub>2</sub>
* Total volume = 100 µL (filled up with 0,5 TBE buffer, 11mM MgCl<sub>2</sub>)
* Controls:
* 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
** DNA [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#MH_255_and_MH_256 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 MH255 without label and MH256_550 labeled with fluorescence donor are used
** Donor only: the strand MH_255 without label and MH_256_550 labeled with fluorescence donor are used
** Acceptor only: the strand MH256 without label and MH255_550 labeled with fluorescence donor are used
** Acceptor only: the strand MH_256 without label and MH_255_550 labeled with fluorescence donor are used
*Concentrations of stock solutions:
*Concentrations of stock solutions:
**Spermin: 50µM and 200µM
**Spermin: 50 µM and 200 µM
**EtBr: 50µM and 200µM
**EtBr: 50 µM and 200 µM
*Concentrations of DNA solutions:
*Concentrations of DNA solutions:
**MH255_647N: 2,8µM
**MH_255_647N: 2,8 µM
**MH256_550: 7,1µM
**MH_256_550: 7,1 µM
-> dilution 1:10 in buffer is used for RtPCR
-> dilution 1:10 in buffer is used for real time PCR
**MH255 (without label for control): 100µM
**MH_255 (without label for control): 100 µM
**MH256 (without label for control): 100µM
**MH_256 (without label for control): 100 µM
-> dilution 1:100 in buffer is used for RtPCR
-> dilution 1:100 in buffer is used for real time PCR
 
Pipeting scheme spermine:


<font size="-1">'''Table XX: Pipeting scheme spermine'''</font>
<center>
{| class="wikitable" border="1"
{| class="wikitable" border="1"
|-
|-
! concentration spermine
! Concentration Spermine
! Spermin Stock [µL]
! Spermine Stock [µL]
! MH255_647 0,28µM [µL]  
! MH255_647 0,28 µM [µL]  
! MH256_550 0,71µM [µL]  
! MH256_550 0,71 µM [µL]  
! buffer (0,5 TBE, 11mM MgCl<sub>2</sub>)
! Buffer (0,5 TBE, 11 mM MgCl<sub>2</sub>)
|-
|-
| 1/19*K<sub>D</sub>
| 1/19*K<sub>D</sub>
| 0,51 (50µM stock)
| 0,51 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 43: Line 46:
|-
|-
| 1/9*K<sub>D</sub>
| 1/9*K<sub>D</sub>
| 1,07 (50µM stock)
| 1,07 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 49: Line 52:
|-
|-
| 1/4*K<sub>D</sub>
| 1/4*K<sub>D</sub>
| 2,4 (50µM stock)
| 2,4 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 55: Line 58:
|-
|-
| 1/2*K<sub>D</sub>
| 1/2*K<sub>D</sub>
| 4,8 (50µM stock)
| 4,8 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 67: Line 70:
|-
|-
| 2*K<sub>D</sub>
| 2*K<sub>D</sub>
| 19,2 (50µM stock)
| 19,2 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 73: Line 76:
|-
|-
| 4*K<sub>D</sub>
| 4*K<sub>D</sub>
| 9,6 (200µM stock)
| 9,6 (200 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 79: Line 82:
|-
|-
| 9*K<sub>D</sub>
| 9*K<sub>D</sub>
| 21,6 (200µM stock)
| 21,6 (200 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 85: Line 88:
|-
|-
| 19*K<sub>D</sub>
| 19*K<sub>D</sub>
| 45,6 (200µM stock)
| 45,6 (200 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
| 49,4
| 49,4
|}
|}
</center>


Pipeting scheme spermine control without labels: Error: there was added 1 µL of buffer too much
<font size="-1">'''Table XX: Pipeting scheme ethidium bromide.'''</font>
 
<center>
{| class="wikitable" border="1"
{| class="wikitable" border="1"
|-
|-
! concentration spermine
! Concentration EtBr
! Spermin Stock [µL]
! MH255 1µM [µL]
! MH256 1µM [µL]
! buffer (0,5 TBE, 11mM MgCl<sub>2</sub>)
|-
| 1/19*K<sub>D</sub>
| 0,51 (50µM stock)
| 1
| 1
| 98,49 (=1µL too much!)
|-
| 1/9*K<sub>D</sub>
| 1,07 (50µM stock)
| 1
| 1
| 97,93 ''
|-
| 1/4*K<sub>D</sub>
| 2,4 (50µM stock)
| 1
| 1
| 96,6 ''
|-
| 1/2*K<sub>D</sub>
| 4,8 (50µM stock)
| 1
| 1
| 94,2 ''
|-
| 1*K<sub>D</sub>
| 9,6 (50µM stock)
| 1
| 1
| 89,4 ''
|-
| 2*K<sub>D</sub>
| 19,2 (50µM stock)
| 1
| 1
| 79,8 ''
|-
| 4*K<sub>D</sub>
| 9,6 (200µM stock)
| 1
| 1
| 89,4 ''
|-
| 9*K<sub>D</sub>
| 21,6 (200µM stock)
| 1
| 1
| 77,4 ''
|-
| 19*K<sub>D</sub>
| 45,6 (200µM stock)
| 1
| 1
| 53,4 ''
|}
 
 
Pipeting scheme Ethidium Bromide:
 
{| class="wikitable" border="1"
|-
! concentration EtBr
! EtBr Stock [µL]
! EtBr Stock [µL]
! MH255_647 0,28µM [µL]  
! MH255_647 0,28 µM [µL]  
! MH256_550 0,71µM [µL]  
! MH256_550 0,71 µM [µL]  
! buffer (0,5 TBE, 11mM MgCl<sub>2</sub>)
! Buffer (0,5 TBE, 11 mM MgCl<sub>2</sub>)
|-
|-
| 0,021*K<sub>D</sub>
| 0,021*K<sub>D</sub>
| 0,51 (50µM stock)
| 0,51 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 174: Line 112:
|-
|-
| 0,044*K<sub>D</sub>
| 0,044*K<sub>D</sub>
| 1,07 (50µM stock)
| 1,07 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 180: Line 118:
|-
|-
| 0,1*K<sub>D</sub>
| 0,1*K<sub>D</sub>
| 2,4 (50µM stock)
| 2,4 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 186: Line 124:
|-
|-
| 0,2*K<sub>D</sub>
| 0,2*K<sub>D</sub>
| 4,8 (50µM stock)
| 4,8 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 192: Line 130:
|-
|-
| 0,4*K<sub>D</sub>
| 0,4*K<sub>D</sub>
| 9,6 (50µM stock)
| 9,6 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 198: Line 136:
|-
|-
| 0,8*K<sub>D</sub>
| 0,8*K<sub>D</sub>
| 19,2 (50µM stock)
| 19,2 (50 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 204: Line 142:
|-
|-
| 1,6*K<sub>D</sub>
| 1,6*K<sub>D</sub>
| 9,6 (200µM stock)
| 9,6 (200 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 210: Line 148:
|-
|-
| 3,6*K<sub>D</sub>
| 3,6*K<sub>D</sub>
| 21,6 (200µM stock)
| 21,6 (200 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
Line 216: Line 154:
|-
|-
| 7,6*K<sub>D</sub>
| 7,6*K<sub>D</sub>
| 45,6 (200µM stock)
| 45,6 (200 µM stock)
| 3,6
| 3,6
| 1,4
| 1,4
| 49,4
| 49,4
|}
|}
 
</center>
Pipeting scheme ethidium bromide control without labels: Error: there was added 1 µL of buffer too much
<font size="-1">'''Table XX: Pipeting scheme ethidium bromide control without labels: Error: there was added 1 µL of buffer too much.'''</font>
 
<center>
{| class="wikitable" border="1"
{| class="wikitable" border="1"
|-
|-
! concentration EtBr
! Concentration EtBr
! EtBr Stock [µL]
! EtBr Stock [µL]
! MH255 1µM [µL]  
! MH_255 1 µM [µL]  
! MH256 1µM [µL]  
! MH_256 1 µM [µL]  
! buffer (0,5 TBE, 11mM MgCl<sub>2</sub>)
! Buffer (0,5 TBE, 11 mM MgCl<sub>2</sub>)
|-
|-
| 0,021*K<sub>D</sub>
| 0,021*K<sub>D</sub>
| 0,51 (50µM stock)
| 0,51 (50 µM stock)
| 1
| 1
| 1
| 1
| 98,49 (=1µL too much!)
| 98,49 (=1 µL too much!)
|-
|-
| 0,044*K<sub>D</sub>
| 0,044*K<sub>D</sub>
| 1,07 (50µM stock)
| 1,07 (50 µM stock)
| 1
| 1
| 1
| 1
Line 245: Line 183:
|-
|-
| 0,1*K<sub>D</sub>
| 0,1*K<sub>D</sub>
| 2,4 (50µM stock)
| 2,4 (50 µM stock)
| 1
| 1
| 1
| 1
Line 251: Line 189:
|-
|-
| 0,2*K<sub>D</sub>
| 0,2*K<sub>D</sub>
| 4,8 (50µM stock)
| 4,8 (50 µM stock)
| 1
| 1
| 1
| 1
Line 257: Line 195:
|-
|-
| 0,4*K<sub>D</sub>
| 0,4*K<sub>D</sub>
| 9,6 (50µM stock)
| 9,6 (50 µM stock)
| 1
| 1
| 1
| 1
Line 263: Line 201:
|-
|-
| 0,8*K<sub>D</sub>
| 0,8*K<sub>D</sub>
| 19,2 (50µM stock)
| 19,2 (50 µM stock)
| 1
| 1
| 1
| 1
Line 269: Line 207:
|-
|-
| 1,6*K<sub>D</sub>
| 1,6*K<sub>D</sub>
| 9,6 (200µM stock)
| 9,6 (200 µM stock)
| 1
| 1
| 1
| 1
Line 275: Line 213:
|-
|-
| 3,6*K<sub>D</sub>
| 3,6*K<sub>D</sub>
| 21,6 (200µM stock)
| 21,6 (200 µM stock)
| 1
| 1
| 1
| 1
Line 281: Line 219:
|-
|-
| 7,6*K<sub>D</sub>
| 7,6*K<sub>D</sub>
| 45,6 (200µM stock)
| 45,6 (200 µM stock)
| 1
| 1
| 1
| 1
| 53,4 ''
| 53,4 ''
|}
|}
</center>


*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. <br>
*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. <br>
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.
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 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)
 
[[Image:FRET efficiency Spermine skal.PNG]] <br>
[[Image:FRET_efficiency_EtBr.PNG]]


<h2>Results</h2>


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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).
|}
<br>
<center>[[Image:FRET efficiency Spermine skal.PNG|x220px]][[Image:FRET_efficiency_EtBr.PNG|x200px]]</center>


__NOTOC__
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1st Sep 2011

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.
  • 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_550 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 XX: 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 XX: 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 XX: 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).

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