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Aga 11-11-2008 11:24 AM

Internal Amplification Control in Real time PCR
 
Forgive me if I missed an identical question here, but I couldn't find it.

I need to establish a TaqMan analysis for quantification of bacteria from various samples without culturing the bacteria on the media.
I've got everything except the internal amplification control (IAC) to check the false negative reactions.

As I understand there are several ways of developing an IAC, mainly the artificial construct cloned in a plasmid, DNA of another organism added to a reaction (together with specific primers and a probe) or a DNA fragment constructed in such a way, that the same primers are utilized for amplification as of a target gene.
My questions are as follows:

1. Which is the best and the quickest way to develop an IAC from your experience?

2. Are there any possibilities of ordering an already constructed, ready-made IAC for applications? If yes, which company offers them? [I have limited time]

3. Is it possible to use IAC in quantification reaction? The co-amplification of another gene will always influence the yield of my target DNA, and in quantification real-time PCR I may not know the concentration of my target DNA so I won'y know the influence of IAC amplification on my target gene!
What's the solution?

Agustin Franco 05-23-2009 02:55 AM

Re: Internal Amplification Control in Real time PCR
 
Quote:

Originally Posted by Aga (Post 155836)
Forgive me if I missed an identical question here, but I couldn't find it.

I need to establish a TaqMan analysis for quantification of bacteria from various samples without culturing the bacteria on the media.
I've got everything except the internal amplification control (IAC) to check the false negative reactions.

As I understand there are several ways of developing an IAC, mainly the artificial construct cloned in a plasmid, DNA of another organism added to a reaction (together with specific primers and a probe) or a DNA fragment constructed in such a way, that the same primers are utilized for amplification as of a target gene.
My questions are as follows:

1. Which is the best and the quickest way to develop an IAC from your experience?

2. Are there any possibilities of ordering an already constructed, ready-made IAC for applications? If yes, which company offers them? [I have limited time]

3. Is it possible to use IAC in quantification reaction? The co-amplification of another gene will always influence the yield of my target DNA, and in quantification real-time PCR I may not know the concentration of my target DNA so I won'y know the influence of IAC amplification on my target gene!
What's the solution?



Hi There:

The problem with selective amplification of one target over another has to do with the design of the primers present in a reaction or the enzyme mix you are using has deficient amounts of taq polymerase. many companies are just too stingy to provide more units of TAQ in a reaction.


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