NB: 260/280 Measurement

Determination of DNA concentration in nexttec 1-Step DNA preparations

We recommend to determine the DNA concentration after purification with nexttecTM 1-Step DNA isolation kits:
 
  • Using the fluorescent dye Picogreen or similar 
 
  • Comparing the fluorescence intensity of DNA bands of unknown concentration with standards, e.g. in Ethidium bromide stained agarose gels
 
  • With restriction: Absorption measurement at 260 nm (A260) in a spectrophotometer (e.g. NanoDrop®)

Background:

The nexttec 1-Step purification system buffer components pass through to the elute. This leads to strong overestimation of the absorption value at 260 nm.
 
There is no influence on downstream applications (PCR, qPCR e.g.), which is shown during inhibiting experiments.
 
The overestimation is relatively constant for similar samples (tissue, blood, plant e.g.).

This allows the calculated DNA at A260 to be corrected bu using the "nexttec Factor".
 
All calculations of the nexttec factor are done in correlation with Picogreen.

Example:
Measured DNA concentration after purification of 10 mg muscle tissue cattle: 160 ng/µl
nexttec factor muscle tissue: 0.3
True concentration: 160 ng/µl * 0.3 = 48 ng/µl

Independent of the A260/A280 ratio (< 1.8) the purified DNA is useable in all downstream applications.
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