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Standard curve - copy number/ul and dilution

viral quantification rt-qpcr sars-cov-2

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#1 Thyago

Thyago

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Posted 12 January 2021 - 11:45 AM

Hello there,

 

I have a question.

 

Assuming I have the following dilution series with known control.

copies/uL 200000 20000 2000 200 20 2

 

My question is, should I add this to my RT-qPCR reaction 4X concentrated to reach the same copies/ul to the advertised or it does not matter?

 

I ask because I was told to use 5 uL from each dilution and my RT-qPCR final volume is 20 uL.

Therefore, if I add 5 uL of the first point, this is 5 x 200 000/copies = 100,000,000 copies in final 20 uL or new concentration of 50,000 copies/uL in the reaction.

 

If I did this way, should I report my results according to 50,000 copies/uL or 200,000 copies/uL?

 

The alternative is to add 5uL of the dilution series 4X as concentrated, so when I add it to my reaction it dilutes and stays at my initial target number.

 

I read a lot of protocols and no one mentions this detail.

 

Thank you very much. 

 

 







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