DNA Quantification
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== Overview == | == 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. | + | 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 work as digesting 500ng of a 4KB plasmid with the same multiple cloning site. | + | *'''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 == | == 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.<br> | |
| + | ::3. Make sure that the A260 measurement is between 0.1 and 1.<br> | ||
| + | :::*If is too low then repeat the measurement using 15X dilution.<br> | ||
| + | ::::*84µl water | ||
| + | ::::*6µl DNA prep | ||
| + | 3. 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)] | ||
| + | 4. Or calculate the μL of dna to add to obtain a desired molar amount of DNA.<br> | ||
| + | *μL = Picomoles*[0.66*DNA Size(bp)]/DNA Concentration(µg/ml) | ||
== Notes == | == Notes == | ||
| + | *One mole of single base pairs weighs 660 grams. | ||
| + | **One picomole of 1000bp weighs 660ng. | ||
==References == | ==References == | ||
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]] | [[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]] | ||
Current revision
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Contents |
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
- If is too low then repeat the measurement using 15X dilution.
- 1. Dilute the DNA sample 30X by combining the following in a cuvette:
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.
