DNA Quantification-S2-PDF

Overview

This protocol uses a spectrophotometer to quantify the amount (μg/mL or ng/μL) of DNA and then uses a simple equation to convert this mass concentration into a molar concentration. The molar concentration is much more useful for most enzymatic processes.

• Example: digesting 500ng of a 2KB plasmid is twice as much “enzymatic work” as digesting 500ng of a 4KB plasmid with the same multiple cloning site.

Procedure

1. Get DNA by any means necessary.
2. Run the DNA quantification (260/280) test on a spectrophotometer.

• Be sure blank a sample first.
• You can use only 1μL of sample if you use a NanoDrop
• Or you can use 90μL of diluted sample using a UV cuvette

1. Dilute the DNA sample 30X by combining the following in a cuvette:

• 87µl water
• 3µl DNA prep

2. Run on the spectrophotometer with a dilution of 30.

3. Make sure that the A260 measurement is between 0.1 and 1.

• If is too low then repeat the measurement using 15X dilution.

• 84µl water
• 6µl DNA prep

3. Calculate the molar concentration of DNA using the following equation:

• Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]

4. Or calculate the μL of dna to add to obtain a desired molar amount of DNA.

• μL = Picomoles*[0.66*DNA Size(bp)]/DNA Concentration(µg/ml)

Notes

• One mole of single base pairs weighs 660 grams.
  • One picomole of 1000bp weighs 660ng.
• 1ug = 1000ng
• 0.001ug = 1ng