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Length of amplicon for RT real time PCR


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#1 Nephrite

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Posted 27 March 2017 - 03:52 AM

Hello!

 

After a long break and a lovable toddler I am back in science - I run my own project smile.png

 

Right now I am trying to pick up my primer pairs for RT real time-PCR and I have a dilemma:

 

In the past I was always performing the RT-reaction on the bench and then proceeding to the real time-PCR.

The primers I designed were  for products 100-120 bp long, for reasons of economy the volume of the reactions was app. 12 microliters - I never had problems.

 

I wonder now, if I increase the volume of the reaction up to 25 microliters (this is the original protocol by the manufacturer of the master mix) could I amplify a bit longer fragments, 150 bp for example?

 

I know that the recommended length is 80-120 bp, but does anybody has an experience with longer fragments in larger volume of reaction?

 

 



#2 bob1

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Posted 27 March 2017 - 05:57 AM

Congratulations on the toddler, and making it back!

 

The length of the fragments is not volume dependent, it is dependent on the polymerase and how long it can extend based on the extension step of the PCR and its attachment to the DNA strand. You should be fine doing longer fragments in the 12 ul reactions. 



#3 Nephrite

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Posted 28 March 2017 - 12:59 AM

Thank you bob1!

I think I will increase the length a bit smile.png






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