For library quantification before sequencing on Illumina, I use the KAPA library quantification kit with their included DNA standards. However, I need to quantify specifically mito or nuclear DNA in steps leading up to this, and I want to make a mito and nuclear specific DNA standard curve. I have specific oligos that I want to use as standards, and I have quantified and made 10X dilution series, just like what KAPA does. Yet my standard curve seems to always be all over the place, while KAPA's is very consistent. Other than the obvious answers like pipet error (we have re-diluted with extreme care multiple times), and freeze thaw problems (each standard is diluted into one-use aliquots and stored at -80), what are some solutions to making a consistent standard curve like the one KAPA puts out?
Alternatively, is there a company that does this kind of thing for you?
Dear Michael
One thing to consider might be the quality of the PCR reaction itself. If the reaction is not properly optimised (and your input template concentration is not standardised) you will struggle to get consistent amplification curves.
I would suggest performing optimising steps using each primer pair ensuring you have the similar and consistent Ct values at a particular template concentration. If you can't obtain this I would look at your template integrity - it's possible that your std curve templates are degraded or contain impurities.
Good luck!
- Badges
- Science topic