DNA Quantification: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
| Line 5: | Line 5: | ||
== Procedure == | == Procedure == | ||
1. Dilute the DNA sample 30X by combining the following in a cuvette: | |||
:*87µl water | :*87µl water | ||
:*3µl DNA prep | :*3µl DNA prep | ||
2. Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30. | |||
3. Make sure that the A260 measurement is between 0.1 and 1. | |||
4. If is too low then repeat the measurement using 15X dilution. | |||
:*84µl water | :*84µl water | ||
:*6µl DNA prep | :*6µl DNA prep | ||
5. Calculate the molar concentration of DNA using the following equation: | |||
<br> | |||
*Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)] | *Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)] | ||
Revision as of 21:47, 29 January 2010
| back to protocols | ||
Overview
This protocol uses a spectrophotometer to quantify the amount (μg/mL) 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 work as digesting 500ng of a 4KB plasmid with the same multiple cloning site.
Procedure
1. Dilute the DNA sample 30X by combining the following in a cuvette:
- 87µl water
- 3µl DNA prep
2. Run the DNA quantification (260/280) test on the spectrophotometer with a dilution of 30. 3. Make sure that the A260 measurement is between 0.1 and 1. 4. If is too low then repeat the measurement using 15X dilution.
- 84µl water
- 6µl DNA prep
5. Calculate the molar concentration of DNA using the following equation:
- Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)]
