DNA concentration ??

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s.shabbidar
s.shabbidar's picture
DNA concentration ??

Hello All
I have a very ?? question about  determination of DNA concentration? In a lab that i work , we dont have nanodrop, so I have to determine it with spect.. but I dont Know HOW?? Please describe me! 
Thanks 

Sami Tuomivaara
Sami Tuomivaara's picture
s.shabbidar,

s.shabbidar,

Zero (blank) the spectrophotometer at 260 nm with the buffer where your DNA is in. Dilute your DNA sample either in water or TE buffer in quartz cuvette and measure absorbance at 260. The absorbance should be in the range of 0.1 to 2 to get most accurate reading, so you might have to adjust the dilution of your sample to get to this range. Repeat the blanking and measurement at 280 nm. Now you have the absorbance of your sample at 260 and 280 nm. Note carefully how much you dilute your original sample, if you dilute 10 ul of your sample with 490 ul of buffer before measurement, the dilution factor is 50.

The equation relating the absorbance to the DNA concentration is:
Absorbance at 260 nm = extinction coefficient at 260 nm * path length (cm) * DNA concentration (ug/mL)
OR
A = e*b*c

for double stranded DNA, the extinction coefficient (e) is 0.02 (ug/mL)-1 cm-1.

Solve for concentration:
c = A/(e*b)

For example, if you use cuvette with path length (b) of 1 cm and your absorbance A260 (A) is 1.5:
c = 1.5/(0.02*1) = 75 ug/mL

This is the concentration of the DNA in the cuvette. If you diluted your original sample before measurement, you have to multiply 75 ug/mL with the dilution factor to get the concentration of your original sample.

Also calculate the A260/A280 ratio, if it is about 1.7 or higher, your DNA is pure, lower ratio means protein contamination.

I'd appreciate if someone checked the math...

Cheers,

s.shabbidar
s.shabbidar's picture
 Suola

 Suola

Thanks for your consideration.

Best,