I want a protocol to measure DNA concentration fluorimetry using ethidium bromide dye.
I do not understand which kind of sample you are measuring. It is the original DNA sample that you want to measure of the PCR product?
Best, I would save 2µl of your product and running a nanodrop.
http://www.thermoscientific.com/content/tfs/en/product/nanodrop-lite-spectrophotometer.html
Else i would still prefer spectrometry.
http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full
Last, but longer and more variable, I would take a stock of DNA with a known concentration and make dilution of it [for example from 100ng/µl to 0.1ng/µl]. Prepare a 1% agarose gel using TBE and add on it 0.5µg/ml EtBr, before it get solid. When solid load the calibration curve, the samples to measure and a marker to estimate DNA size. Run it in TBE and detect it using UV light and camera.
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
Regards
no I wanna measure encapsulation effeciency of my nanoparticles so I want to measure non binding DNA in suernatant which of very minuit ammount about 0.5ng\ul which in error range of nanodrop
We are using both picogreen
http://www.promega.com/~/media/files/products%20and%20services/instruments/detection/tbs%20technical%20support%20docs/s-0109.pdf
or bioanalyzer high sensitivity
http://www.genomics.agilent.com/en/Bioanalyzer-DNA-RNA-Kits/High-Sensitivity-DNA-Analysis-Kits/?cid=AG-PT-105&tabId=AG-PR-1069
regards
use EtBr and read the sample and std curves with a Victor plate reader (perkin elmer) or similar.
Sorry I have no experience with the EtBt instead of picogreen. I found papers in which they claim that EtBr has a sensitivity of 2-25ng and less accurate that picogreen
http://nar.oxfordjournals.org/content/24/13/2623.full
http://www.phenixresearch.com/Images/TN_FluorecentProbeQuantitation.pdf
Have you seen this protocol?
http://www.molvis.org/molvis/v8/a49/v8a49-rengarajan.pdf
I use EtBr fluorometry for relative quantification but not for absolute quantification. It seems to work fine for this purpose using the following method & condition:
- the concentration of DNA I use (=/< 4ng/ul DNA, 40uL per sample),
prepare 'blank' samples (i.e. buffer alone) and dilution series of your 'input' DNA to make a standard curve. Better to prepare samples in duplicates - triplicates.
- place 40uL DNA and blank samples into wells of a 96-well optical plate.
- mix with the equal volume of 0.5ug/mL EtBr solution.
- incubate for 10 - 15 min at RT.
- measure fluorescence using a plate reader (I use Tecan plate reader using the setting: excitation = 360 +/- 5nm, emission =612 +/-5nm).
- use excel to plot a standard curve & calculate the DNA concentration of your sample (do not forget to subtract the value of the blank sample from the values of the DNA samples).
Note that EtBr gives quite a bit of background signal and as your starting DNA concentration is very low, it might be difficult to differentiate the actual signal from the 'noise'. Maybe you can play with the concentration of EtBr to get an optimal signal to noise ratio.
For the concentration range you are testing, I think it is better to use picogreen or SYBR Green I instead of EtBr for accurate and reproducible results though...
my sample concentration ranged from 400 ng/ml to 1000ng/ml do u think that ethidium bromide cant be used
which sybr green product can i buy ffor that and from which compANYbe more economic
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