Which is the best way of determining the concentration of DNA? - (Apr/15/2008 )
Dear forumers,
Which is the best way of determining the DNA concentration of a PCR product
1) OD260
or by comparing a target band with a band which has a known concentration on agarose gels?
Thank You in advance
Which is the best way of determining the DNA concentration of a PCR product
1) OD260
or by comparing a target band with a band which has a known concentration on agarose gels?
Thank You in advance
Likely the conc of the PCR is too high to measure by OD @ 260 nm.
Make a dilution series (1:10) and measure multiple samples, then back calculate.
This is assuming you have purified the product from a single band in a gel to remove the primers, etc.
Also, to determine the copy number, use the length of the PCR product and plug it into the following equation:
number of copies = ( amount * 6.022x10^23) / (length * 1x10^9 * 650)
or:
number = (ng * number/mole) / (bp * ng/g * g/ mole of bp)
or take the short cut and go here... http://www.uri.edu/research/gsc/resources/cndna.html
Cheers,
Hope that helps
For PCR products I usually compare it with some other band of known weight. It's not very acurate, but for most applications it's more than enough.
To do OD600, like rockymtnlab said, you need to purify your product anyway so you won't have interference from primers and so on.
nanodrop, a spectrophotometer is most accurate. But as other have mentioned your DNA sample must be clean. The spectrophotometer measures the total nucleic acid concentration in the sample. Contamination with fragmented DNA and RNA will throw off the estimated concentrations of your sample DNA.
Comparing your DNA sample to a quantified DNA ladder is better in that regard, but it isn't very accurate. It is part guess work trying to determine how bright is your sample to the brightness a rung in ladder.
Personally I prefer the Nanodrop. It is faster than running a gel. And with practice, RNAse and PEG precipitation you can get a clean plasmid sample. A labmate and I also did a test, we found that our estimates of DNA concentration by comparison with a quantified DNA ladder was almost the same as the value given by nanodrop. So guesstimation was almost the same as accuracy with a little contamination, which meant speed decided the winning method.