High concentration DNA on spectrophotometer but no band on agarose gel. WHere is - DNA from bone and teeth (May/01/2008 )
Hi,
I extraction DNA from bone and teeth by silica method. I used DNAzol as a chaotropic salt. Why I have high concentration DNA (cca 420 ng/ul from bone) after extraction of DNA with this method. The ratio of 260/280 nm was 1.17 - 1.47.
But I used an agarose gel and I did not find any band of DNA at agarose gel. I used PCR for detection of species (cow) and I became five PCR product (good quality) from seven DNA samples. But I do not understand, why my spectrophotometer give me high value of concentration of DNA and agarose gel do not shown the presence of DNA.
Thank you
miso
I extraction DNA from bone and teeth by silica method. I used DNAzol as a chaotropic salt. Why I have high concentration DNA (cca 420 ng/ul from bone) after extraction of DNA with this method. The ratio of 260/280 nm was 1.17 - 1.47.
But I used an agarose gel and I did not find any band of DNA at agarose gel. I used PCR for detection of species (cow) and I became five PCR product (good quality) from seven DNA samples. But I do not understand, why my spectrophotometer give me high value of concentration of DNA and agarose gel do not shown the presence of DNA.
Thank you
miso
Perhaps it's a smear of different sized DNA fragments on the whole lane, that altogether result in a high concentration and the sequences you need are anyway unbroken.
I extraction DNA from bone and teeth by silica method. I used DNAzol as a chaotropic salt. Why I have high concentration DNA (cca 420 ng/ul from bone) after extraction of DNA with this method. The ratio of 260/280 nm was 1.17 - 1.47.
But I used an agarose gel and I did not find any band of DNA at agarose gel. I used PCR for detection of species (cow) and I became five PCR product (good quality) from seven DNA samples. But I do not understand, why my spectrophotometer give me high value of concentration of DNA and agarose gel do not shown the presence of DNA.
Thank you
miso
1.17-1.47 is not a very good ratio. you may have a relatively high amount of protein contamination. your dna concentration is probably a lot lower than you think.
Hi mdFenko,
I know, that my concentration of DNA is not high but my spetrophotometer say that is very high and the agarose gel say that I have not DNA from DNA and Teeth but my PCR reaction was good. I want to know why ma spectrophotometer make mistake. And the ratio 1.17 -1.47 does not mean that the PCR does not work.
Your sample is loaded with protein which is giving you a false reading in the spectrometer and is why you have such a high ratio. The spectrometer is indicating you have high DNA content but much of the absorption is coming from the proteins, thereby giving you a high reading. Your sample actually has much less DNA but PCR is incredibly sensitive and only requires very, very small amounts of DNA in order to get a good reaction, hence why your PCR is fine. However, agarose gels are based on visual detection and therefore are much less sensitive. It requires much more DNA to be able to see it in a gel than it takes to get a PCR to work. Run out as much of the DNA sample as you can spare, soak the gel in EtBr for at least 5-10 mins and then destain until the gel itself no longer gives any signal under UV. You might be able to see a DNA band then. If you want to get rid of contaminating proteins and obtain a much more accurate spectrometry reading, you need to phenol/chloroform extract the DNA followed by EtOH precipitation.
Hi rkay477
Thank for your explanation. Its very usefull for me. Thank you very much.