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

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Pipeting scheme:
Pipeting scheme:


{| class="wikitable"
{| class="wikitable" border="1"
|-
|-
! concentration spermine
! concentration spermine
! header 2
! Spermin Stock [µL]
! header 3
! MH255_647[µL]
 
! MH256_550[µL]
 
! buffer
|-
|-
| row 1, 1/19
| 1/19*K<sub>D</sub>
| row 1, cell 2
| 0,51
| row 1, cell 3
| 3,6
| 1,4
| 94,49
|-
|-
| row 2, 1/9
| 1/9*K<sub>D</sub>
| row 2, cell 2
| 1,07
| row 2, cell 3
| 3,6
| 1,4
| 93,93
|-
| 1/4*K<sub>D</sub>
| 2,4
| 3,6
| 1,4
| 92,6
|-
| 1/2*K<sub>D</sub>
| 4,8
| 3,6
| 1,4
| 90,2
|}
|}


Revision as of 05:47, 2 September 2011

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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:

concentration spermine Spermin Stock [µL] MH255_647[µL] MH256_550[µL] buffer
1/19*KD 0,51 3,6 1,4 94,49
1/9*KD 1,07 3,6 1,4 93,93
1/4*KD 2,4 3,6 1,4 92,6
1/2*KD 4,8 3,6 1,4 90,2


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