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Standard curve using ImageJ

strandard curve dna amount imagej

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#1 CristianaCris

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Posted 14 June 2016 - 08:30 AM

Hy,

 

I want to make a standard curve using the intensity/area of DNA Ladder by ImageJ for measuring the amount of dna.

I measured the intensity of ladder's bands (the image from the producer) and I made a standard curve. Now I want to correlate the intensity of dna from the gel.

 

I don't know if my approach is the best or correct and I would like an opinion from you.

 

Thank you!



#2 bob1

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Posted 14 June 2016 - 01:28 PM

Your general approach is correct, but you must load standards with every gel and use those to generate a standard curve each time. You can not use images from previous gels (or the ones provided by the producer) and different exposures as these will lead to erroneous results.

 

Bottom line: each time you run a gel, load the standards as well and use those.



#3 CristianaCris

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Posted 17 June 2016 - 10:30 AM

Thank you!

 

I run the samples with 2 microliters dna marker, but the image from the producer was obtained by using 1 microliter. In order to get the standard curve can I double the concentrations of the marker. If 500 bp is coresponding to 73 ng (1 microliter) can I consider for 2 microliter 146 ng?



#4 bob1

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Posted 17 June 2016 - 12:05 PM

Yes. In fact you can apply this to any volume you choose. Note that to obtain an accurate standard curve your sample should be about the same length as the standard you are comparing to as length and brightness can be correlated for these sorts of things.

#5 CristianaCris

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Posted 08 July 2016 - 08:46 AM

Thank you very much for your help!







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