Hi everbody. I have been studying human mitochondrial DNA and cell free nuclear DNA in whole blood using mutliplex real time PCR. I just can't make standard curve because I can't make plasmid as some papers may show. Other papers mention to measure DNA extract in control samples and make serial dilutions and measure it by nanodrop to obtain the curve. Others say to get the patient sample with the lowest CT being of highest concentration for each marker and make serial dilutions and measure it by nanodrop. How would I proceed?? thanks in advance
HI
The simplest and cheap solution is:
https://eu.idtdna.com/pages/products/genes/gblocks-gene-fragments
make your dDNA standard with synthetic DNA
best regards
For human you just choose a house keeping gene e.g. growth hormone to make a serial dilution.
The last variant may be suitable as well as using purified DNA with known concentration from any human cell culture. But the ratio of nuclear to mitochondrial DNA may differ and if you need to know exact copy numer, try digital PCR or use synthetic standards. Try to design the shortest possible amplicon because cell-free DNA may be sufficiently degraded
- Badges
- Science topic