DNA concentrations

[John D]

Hi

hoping someone can assist - lawyer working on DNA case

If the extracted DNA is .25 nanograms per microlitre how would the biologist achieve .5 nanograms per microliter for purposes of then amplifying with the amplification kit???

Is it as simple as adding 2 microlitres to the solution???? I don't get how that increased the amount of DNA to be amplified as the concentration seems to remain the same??

Thanks

John

[Ola]

Hi,

adding a larger volume would make it easier to amplify even though the concentration is the same since you then will have more template molecules. It is also possible to amplify from less than 0.5 nanogram (one copy of the human genome weighs ~0.006 ng).

[nucacidhunter]

To double the contration, volume of DNA solution need to be halfed. Three options:

1- dry down the solution with speedyvac or similar device. This will increase the salts concentration which might be detrimental for some downstream applications.

2- using clean up columns or beads in which DNA binds to a solid support and then eluting bound DNA with less volume of buffer. Some DNA will be lost in the process.

3- using Amicon Ultra Centrifugal filters which allows liquid and small molecules to pass through a filter and DNA stays behind.

[seqtechno1]

concentrating DNA

Do a concentration and purification using AMPure XP magnetic beads (they are SPRI beads, you can google how they work if interested) and concentrate and purify the DNA using these and a magnetic stand/rack. The trick is you will also need some sort of re suspension buffer which typically contains PEG I think? Its hard to tell b/c most of them are proprietary and come in disposable kits used for sequencing.