Biomod/2011/TUM/TNT/LabbookA/2011/09/01: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Silvia Blank (talk | contribs) |
Manuel Hora (talk | contribs) |
||
Line 8: | Line 8: | ||
==Real time PCR with MH255_647 and MH256_550 adding ErBr and Spermine== | ==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. | * 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 | * The DNA intercalator ethidium bromide and the major groove binder Spermine are added at various concentrations according to their K<sub>D</sub>-Values. | ||
* The K<sub>D</sub> value of Spermin is 4,8µL and that of ethidium bromide is 12µL. | * The K<sub>D</sub> value of Spermin is 4,8µL and that of ethidium bromide is 12µL. | ||
* The concentrations of Spermin and ethidium bromide are as follows: | * The concentrations of Spermin and ethidium bromide are as follows: | ||
Line 291: | Line 291: | ||
|} | |} | ||
*For hybridisation of DNA helix, the samples are incubated at 65 °C. The temperature is lowered by | *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. | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 02:11, 7 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> </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
-> dilution 1:10 in buffer is used for RtPCR
-> dilution 1:100 in buffer is used for RtPCR Pipeting scheme spermine:
Pipeting scheme spermine control without labels: Error: there was added 1 µL of buffer too much
Pipeting scheme ethidium bromide control without labels: Error: there was added 1 µL of buffer too much
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. |