Real time PCR of mtDNA

Amplification curves of mtDNA in either 10µl (n=48,± 1std) or 20µl (n=24,±1std) reaction volume. A 1000µl in-house mix was distributed into a 96 well plate (VWR, European Cat. No. 211-0297) and analysed in a CFX Connect Real-Time PCR System. The cycling conditon was the following; Initial denaturation,  2min @94°C, 45 cycles of, 20sec @94°C, 20sec @57°C, 40sec @72°C and reading plate. Both FAM (Eva green) and Hex (TaqMAN probe) channels were recorded.

PCR mix 

Components marked with yellow are produced or formulated in the lab.

 

Average Cq values by regression.

 

 

Amplification curves (realtime PCR)

 

Amplification of nuclear and mitochondrial specific primer by use of in-house chemistry. The items marked with yellow is made in the lab. Primer, dye and dNTP are purchased from commercial sources. PCR volume was 20µl with 24 technical replicates for each amplicon.

Recipe

Cycling

  Cq 1 STD
Mitochondrial DNA 13,96 0,04
Nuclear DNA 20,62 0,08

 

 

 
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