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

From OpenWetWare
Jump to navigationJump to search
Line 52: Line 52:
| 1,4
| 1,4
| 90,2
| 90,2
|-
| 1*K<sub>D</sub>
| 9,6
| 3,6
| 1,4
| 85,4
|-
| 2*K<sub>D</sub>
| 19,2
| 3,6
| 1,4
| 75,8
|}
|}


Revision as of 05:49, 2 September 2011

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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
1*KD 9,6 3,6 1,4 85,4
2*KD 19,2 3,6 1,4 75,8


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2011/TUM/TNT/LabbookA/2011/09/01&oldid=533531"