Question

Can you pleasee answer these questions regarding the experiment of DNA quantification. What is “blank” and why do we use it in DNA quantification? If you are told to analyze DNA samples that we extracted from buccal swabs, what would you use as a blank?
3. Explain in details:
a. Concentration
b. A260/A280 ratio
c. A260/A230
Why are those measurements used, how do you know which concentration to expect if
you used the commercial kit for DNA extraction, what is approximate range for b) and c)

Thank you in advance. You do not know how much I appreciate your help!!

Answer 1

Blank is basically the solvent in which you dissolve your sample like if your are collecting sample from buccal swab then you should resuspend it in saline (0.85%NaCl),so your saline become blank

Blank always included in spectrophotometer reading as we assume that light which instruments pass to know concentration is absorbed partially by solvent and solute or sample both, but you want to know concentration of solute only and hence we minus light absorbed by solvent and blank is very important.

1st we take reading of blank sample and then do zero and then start taking samples reading.

3)a concentration is amount of solute present in given solution/ solvent like DNA is resuspended in 100 ul of distilled water then the amount of DNA is water is known as concentration.

B) 260/280 used for assesing purity of DNA with protein as DNA showed absorbance at 260 and protein at 280 if reading of 260/280 is below than 1.8 then DNA is contaminated with protein.

C)Ratio 260/230 indicated that DNA is contaminated with phenol or salty .it's value should be more than 1.8

For commercial kit for DNA extraction 260/280 is more than 1.8 while 260/230 should be more than 1.5.

Hope it's clear..thanks