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How to optimize a master mix kit?


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#1 kabtq9s

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Posted 11 March 2015 - 10:18 AM

So according to the below paragraph, I need to sequentially increase the concentration of the components. But how do I do that if I am using an already made maser mix kit with fixed concentrations from the company. I don't think we can even find out what concentrations are used for each reagent ?

 

Multiplex qPCR Assay Optimization

If a multiplex reaction is not optimized, the amplification of a more efficient or more abundant target can inhibit that of a less efficient or less abundant target. This occurs because reaction components such as DNA polymerase, dNTPs, and MgCl2 become limiting in later cycles, and the amplification of the less efficient or less abundant target is compromised. Inhibition of a target's amplification will cause its Cq to be delayed in a multiplex qPCR assay relative to that in the singleplex assay. One method for optimizing multiplex reactions involves sequentially increasing the concentrations of DNA polymerase, dNTPs, and MgCl2.

 

 

Thank you 


Edited by kabtq9s, 11 March 2015 - 10:20 AM.


#2 hobglobin

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Posted 11 March 2015 - 10:39 AM

For multiplex reactions, especially the primer concentrations should be optimised at least for endpoint PCRs.


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#3 kabtq9s

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Posted 11 March 2015 - 10:57 AM

Thanks hobglobin! 

 

So I guess there is no way to optimize the reagents in the pre-made master mix? just the primers ?



#4 hobglobin

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Posted 11 March 2015 - 11:31 AM

Not much idea about realtime PCR, but I guess there are extra kits for this and protocols how to optimise such reactions. Depends surely also which kind of PCR, and how it is quantified (dyes, probes)...

Anyway if you want to change concentrations of other ingredients (esp. Mg2+) you need the original concentration, otherwise it's "blind flying". Try to get information from internet, or ask the company.


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.


#5 phage434

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Posted 11 March 2015 - 02:26 PM

The Mg++ concentration will be in the 1.5-2.5 mM region. You can increase the enzyme, dNTP, and Mg++ concentrations (but not the relative amounts) by simply using a higher amount of the master mix in the reaction.  If the bottle says it is a 2x, instead of adding water to make up the extra volume, leave some of the water out, so that the dilution is 1.9 to 1.7x.



#6 kabtq9s

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Posted 12 March 2015 - 08:07 AM

Thanks Phage,

 

but just to make sure, so if this is my intended reaction are you saying that I should use 11 ul instead of 10 ul for the master mix and increase the water to 4 ul instead of 3 ul ?

 

capture1.jpg



#7 phage434

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Posted 12 March 2015 - 10:05 AM

No, at the end, the amount of 2x master mix should go up and the water stay the same or go down. This will increase the concentration of enzyme, dNTPs, and buffer (including the magnesium) in the final reaction.



#8 Trof

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Posted 19 March 2015 - 06:04 AM

kabtq9s, you didn't write what your problem was in the first place.

No amplification? Faint amplification? Uneven yields from smaller-bigger products? Unspecifities?

Depending on that you should decide optimization strategy.


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