DNA Quantification: Difference between revisions
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(New page: {{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...) |
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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. 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. | 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 == | == Procedure == | ||
1. Dilute the DNA sample 30X by combining the following in a cuvette | 1. Get DNA by any means necessary. | ||
<br> | |||
2. Run the DNA quantification (260/280) test on a spectrophotometer. | |||
2. Run | *Be sure blank a sample first. | ||
3. Make sure that the A260 measurement is between 0.1 and 1. | *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 | |||
Picomoles/µl = DNA Concentration(µg/ml) / [0.66*DNA Size(bp)] | ::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)] | |||
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. | |||
* 1ug = 1000ng | |||
* 0.001ug = 1ng | |||
==References == | ==References == | ||
"Convert ng to ug - Conversion of Measurement Units." http://www.convertunits.com/from/ng/to/ug | |||
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]] | [[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]] | ||
Latest revision as of 10:06, 14 March 2014
| back to protocols | ||
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.
- 1ug = 1000ng
- 0.001ug = 1ng
References
"Convert ng to ug - Conversion of Measurement Units." http://www.convertunits.com/from/ng/to/ug
