dsDNA quantitation via Realtime-Cycler and SybrGreenI - (Jan/18/2006 )
In the paper JM Gonzalez et al "Optical Thermal Cycler for Use as a Fluorometric Plate Reader to Estimate DNA Concentrations", BioTechniques 34, pp 710-712 (April 2003) a method was described to quantize dsDNA concentrations via a Realtime Cycler (iQ iCycler from BioRad). I tried that method on our ABI7500 system and I used dilutions of calfthymus DNA for standard
curve points. However, I was not successful, I was not able to create a standardcurve.
I tried once only with SybrGreenI (final conc. 1:80000 and 1:160000) and once with SybrGreenI and ROX as a passive reference.
final DNA-conc. in tube : 40ng/µl; 16ng/µl; 8ng/µl; 4ng/µl; 0,8ng/µl and 0,08ng/µl
In both measurements values varied too much. Did anyone else try this method and what are
your experiences ?
Thx in advance for your input,
I have tried a similar method using picogreen on the ABI 7700 using a single read. I have had good luck with the standard curve so far, however some of my sample readings have been off. Still trying to figure out if their might be anything inhibiting the readings The protocol was adapted from this paper