I have DNA extracts of bacterial strains (>300). I am having trouble quantifying the DNA accurately. I have done nanodrop, qubit, quantit and agarose gels to test the quality and quantity of DNA. I am certain that I have enough high molecular weight DNA from the gels and I can see that the nanodrop contamination ratios are not too bad. However, the go to standard fluorescence based measurements that I did high-throughput quantit and qubit but I can see very high variation between repeats in quantit as well as the qubit repeat. The only reason I could think of is clumps of undissolved DNA in the buffer and I am pipetting incorrectly between reps. I have read about heating but I don't want to damage the DNA as the buffer I am using has low DNAse blocker (0.1mM EDTA). I would love to hear some ideas as to what is going on and how to solve it.
Do the calibration curve/standard curve again and then take the readings of sample. Make sure that the dye used in not expiry one. Try using fresh dye.
Article Pitfalls of DNA Quantification Using DNA-Binding Fluorescent...
I'm not sure what you mean by "clumps of undissolved DNA. DNA would remain aqueous in these cases. Did you vortex your samples thoroughly?
Redo your standard curve and check accuracy with any commercial plasmid with known concentration. Also, for Qubit prepare a master mix and then prepare your samples, do not forget vortexing and the two minutes waiting period before each reading. Hope it works!!
In our lab we tried to go to standard fluorescence based measurements and always found a big difference to the Nanodrop results. Now we are back to only Nanodrop. We tried many things and could not find out the reason for the difference.
Do you use a heated elution step when purifying the DNA? The Qubit dye intercalates specifically into double-stranded DNA. Heated elutions can sometimes cause random regions of single-stranded DNA to form, which will affect the accuracy of Qubit measurements (and will vary from sample to sample). If you're worried, try denaturing the sample completely by mixing 1:1 with 0.5mM NaOH and measuring again on a Nanodrop, using the single-stranded DNA module (DNA-33).
- Badges
- Science method