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| Hey guys! First, i want to tell you sorry for my bad english there is my problem: So, i need to make Master Mix =) what a differense from basic PCR? I mean...eeemmm...Do anybody speak Russion? Eh.. Do you know any links for literature about Master Mix for Real-Time PCR? Smth like: Total concentration for reagents (total volume 50 ml): DNA ~ 100-400 ng per reaction Tag ~ 1 unit/ml ~ 1 ml per reaction Primer A, Primer B ~ 1 ml per reaction (0.2 pMol) 10pMol/ml in Stock Probe ~ MgCl2 ~ 2-2.5 mM per reaction dNTP ~ 400 mM per reaction Rox ~ Do i need Rox for detect Luminescence? What concentration of Probe do i need for reaction? Unfortunately all of protocols ive seen, autors just wrote "WasterMix" without detalisation. 10x =) |
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| Hey there Leowe! A real-time PCR reaction mixture can be either 50 ml or 25 ml. Prepare the following mixture in each optical tube. 25 ml SYBR Green Mix (2x) 0.5 ml liver cDNA 2 ml primer pair mix (5 pmol/ml each primer) 22.5 ml H2O OR 12.5 ml SYBR Green Mix (2x) 0.2 ml liver cDNA 1 ml primer pair mix (5 pmol/ml each primer) 11.3 ml H2O http://pga.mgh.harvard.edu/primerbank/PCR_protocol.html Also In addition to the use of ROX, a master mix should be used when setting up multiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility (ROX will be within the master mix). http://www.dorak.info/genetics/realtime.html Please let us know your successes and failures! Hope you get many success. |
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Thx for reply! Is there any differense between MasterMix for Real-time PCR and Reverse Transcription PCR? may i follow M.Tevfik Dorak's guide? As i know, he wrote recomendations for Real-Time RT-PCR. Eh, sorry if my questions are quite stupid ![]() eeee...i mean differense in common reagents =) not Reverse Transcription components Last edited by Leowe; 11-20-2006 at 07:02 AM. |
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| I bought a ready made master mix from applied biosystems for taqman (its the easiest way, since you are doing taqman)... Also for RT-PCR ( i do 2 step real time RT PCR) it is easier to use a kit since you need pcr buffer, MgCl2, reverse transcriptase, RNAse inhibitor and primers :S Its quite many things to find out...
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