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real-time pcr non reproducible

real-time per

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#1 alice.lunardon

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Posted 20 August 2013 - 06:50 AM

Hello,

compliments for the blog, I am new of it.

 

I have a very difficult problem to solve with real-time pcrs: I am using dilutions of cDNA pools to test for primer efficiency. The same day I make new dilutions (1:10, 1:50, 1:100,..) I obtain a good standard curve; when I freeze the dilutions and I repeat the same reactions the day after I obtain higher cycles for each dilutions, and they increase if I freeze again my dilutions and repeat the reaction for a third time. It seems that more time passes more my cDNA degrades. The least diluted cDNA (1:10) is the one that is more stable over repetitions. 

 

I use DNase-free RNase-free water and filter tips. The negative control is clean.

 

How can I know if I have contamination in my original RNA or cDNA? 

 

Please if you have any suggestions they would be of great help for me!

 

Thank you!

 

Alice



#2 bob1

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Posted 20 August 2013 - 12:39 PM

Freeze-thawing damages DNA by shearing of the DNA.  There isn't any easy way to prevent this, but storage at 4 C will in TE (or 10 mM tris pH 7.5) will work well for most DNA.



#3 phage434

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Posted 20 August 2013 - 01:09 PM

Yes! Store in TE, even at room temperature, but better at 4C.



#4 alice.lunardon

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Posted 20 August 2013 - 02:38 PM

Thanks for the answers; I read that salts present in the buffers can cause problems during real-time pcrs, in fact people in my lab store cDNA in water at -20°C and use it after 10 times of freeze and thawing without any problems, so I think that the problem could be in the starting material: do you think that there could be some inhibitors or degrading enzymes in the original RNA or introduced in the reverse transcription phase that increase their action after thawing?

 

Thank you very much!!

 

Alice



#5 phage434

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Posted 20 August 2013 - 05:46 PM

Use arithmetic. With TE, there is 1 mM EDTA. It will chelate magnesium, protecting your DNA from DNAses. Your template in the PCR reaction is about 0.1 of the volume, so the PCR reaction will have 0.1 mM EDTA. Now look at the PCR buffer magnesium concentration. Typically 1.5 - 3 mM. Now tell me that this makes a difference.

Rumors spread by people who can't do arithmetic (there are a lot of them).






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