Quant-iT assay development not returning correct values - (Jun/19/2013 )
I am trying to implement the Quant-iT dsDNA high sensitivity assay in a 384 plate format. According to the manual, the normal set up is in a 1 mL assay volume, and involves diluting the dsDNA dye 1/200 in 500 uL before adding 500 uL of sample or standard. I have been diluting the dye 1/20, and mixing 15 uL of diluted dye with 10 uL of dsDNA standard or sample in each well. After some incubation time, I then use a plate reader to read the fluorescence values. I have been able to get a linear standard curve (0 to 100 ng); however, whenever I try to run a PCR product (adding 10 uL of diluted PCR product to 15 uL diluted dye), it does not give me the 'correct' value. I have been setting up all samples/standards in triplicate, and I also run water as a negative control.
I base the correct value on the number that the Qubit gives me, using the protocol as described in their manual. I am wondering if anyone else has tried to use a dye based DNA quantification assay in a small volume (~25 uL) format and if similar issues were encountered. In particular, my Quant-iT values are always lower than the Qubit values (the factor varies, but it differs as much as two fold).
Thanks for the help!
PS I am new here and also new to the field
First I thought the kit is only made for Qubit readers but it's not. But perhaps it's an inaccuracy of the plate reader? And another factor might be dNTPs in the PCR product which are either measured or not depending on method (not sure)?
Anyway I'd try out another fluorometer...