Hi,
I need help with a method to quantify DNA in ng/ul for sequencing. I don't have access to Qubit or Nanodrop instruments and want to use a UV spectrophotometer. Is this possible and accurate?
Could someone help with a detailed protocol please?
Hello,
Yes, the quantity and purity of nucleic acids can be determined by spectrophotometry. Nucleic acids have an absorption maximum at 260 nm.
A solution that has 50 µg/mL of double-stranded DNA has an absorption of 1 at 260 nm.
You dilute your DNA solution and measure its OD at 260 nm, what you find as OD, you convert it using the rule 1OD = 50 µg/mL.
Next, don't forget to calculate the initial concentration taking into consideration the dilution factor.
If you need the explanation for determining the purity of your solution by spectrophotometer, tell me.
Thanks so much Sofiane for your reply.
Yes, I need the protocol for determining purity please.
Hello,
Calculate the 260 nm/280 nm ratio, a ratio less than 1.8 means your sample is contaminated with proteins.
Between 1.8 and 2 means your sample is pure and not contaminated with proteins.
same thing for determining contamination with polyphenols/polysaccharides or not, but this time calculate the 260nm/230nm ratio.
@sofiane Thanks Sofiane! That was very helpful.
Do you recommend a specific spectrophotometer? I'm in the process of buying a new one. Just can't afford the expensive ones I mentioned above.
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