T Nation

Attn Biochem Nerds

Really off topic, but I need help with converting something for my lab writeup. I just can’t seem to calculate this without seeing an example of it.

Q: Calculate the concentration (mg/mL) of DNA using an extinction coefficient of 250 (for a 1% solution (weight/volume), a light path of 1.0cm, and a wavelength of 260.

A: Absorbance at 260 was 0.657, and the equation is:
C=Abs/ext coeff*pathlength

Seems easy enough, but the answer comes out in mol/L, I got 0.002628 mol/L, and now need to convert it to mg/mL. I know this has something to do with the 1% solution, but i don’t know how to do it. Anyone else know???

Beer’s lay in general isn’t very useful for determining DNA concentrations, as the molar extinction coefficient will vary based on the CG and AT content of your DNA and if it is single stranded or double stranded. If you can do the readings again, your spectrophotometer should give you the option of using the A260/A280 ratio to determine the concentration of the DNA. This method will be a lot easier and more accurate.

Sorry i didn’t even see the last paragraph in your post. If you know how many base pairs long your DNA stand is you can use the average base pair weight. It is something around 600 Daltons. Use that to estimate how many Da your strand is, then multiply by the molar concentration to get how many grams per liter there are. To convert to mg/mL you would divide by 1000 and multiply by 1000 so the number should be the same.

Although i still stand by the A260/A280 ratio as the better method.

[quote]Rah-Knee wrote:
Beer’s lay in general isn’t very useful for determining DNA concentrations, as the molar extinction coefficient will vary based on the CG and AT content of your DNA and if it is single stranded or double stranded. If you can do the readings again, your spectrophotometer should give you the option of using the A260/A280 ratio to determine the concentration of the DNA. This method will be a lot easier and more accurate.[/quote]

You can travel through time?

My A260/A280 ratio was 0.9 or so, which is pretty impure I guess, plus I don’t know how many bp the DNA is…so I guess that method is out.

I think I found a reasonable quation on some website though. Not sure how they came up with it though seeing how C=Abs/ext. coeff*pathlength.

They postulate that:

?Concentration = Absorbance x Extinction coefficient x dilution factor (DF) x path length
? So by using an extinction coefficient of 250, absorbance of 0.657, dilution factor of 0.01(0.1/10), and path length of 1, we get:
?C = 0.6572500.01*1
?C= 1.64 mg/mL.

Does thi equation make sense?