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  2. → Viewing Profile: Posts: thekid

thekid

Member Since 04 Feb 2009
Offline Last Active May 27 2013 08:29 PM
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Posts I've Made

In Topic: Normalization of Real time PCR results using Pfaffl method

06 August 2012 - 12:51 PM

Hello,

   AM planning to do expression studies too and would like to use the pfaffl method for data analysis. But I am doing my experiments on a ABI 7500 machine which calculates based on the delta delta ct method. Woul like to know if I can calculate the pfaffl method using the raw data. I did come across a program called Qbase plus but it is a paid software. Would like to know if there is a free software that can be used to calculate the pfaffl method using the raw data.


regards,
thekid

In Topic: Taqman Probes problem

02 August 2012 - 10:54 AM

I agree that the limitations are 20mer's but I have worked with probes that are upto 25mers. What is more worse in the current situation is that I have reduced the anneal all the way to 50C and still there is no detection in the amplification plot. I noticed that in the multicomponent plot that the Tamra has a higher signal followed by Rox and Fam this I would assume is because the Probe has not bound to the plasmid and hence Fam signal is still being absorbed by Tamra and hence more of Tamra signal than Fam.

But I have already lowered the anneal to as low as 50C.

thekid

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