|Notes on handling DNA|
|Nature of DNA|
|The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation. Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments, high temperature, and UV irradiation. Careful isolation and handling of high molecular weight DNA is necessary to ensure that it can be used well in various downstream applications. Damaged DNA could perform poorly in applications such as genomic Southern blotting, long-template PCR, and construction of cosmid libraries.|
|Handling fresh and stored material before the extraction of DNA|
|For the isolation of genomic DNA from cells or tissues, use either fresh samples or samples that have been rapidly frozen in liquid nitrogen and stored at - 70°C. This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases.|
|Storage of DNA|
|Store genomic DNA at +2 to +8°C. Storing genomic DNA at -15 to -25°C can cause shearing of DNA, particularly if the DNA is exposed to repeated freeze-thaw cycles. Plasmid DNA and other small circular DNAs can be stored at +2 to +8°C or at -15 to -25°C.|
|Drying, dissolving and pipetting DNA|
|Avoid overdrying genomic DNA after ethanol precipitation. It is better to let it air dry than to use a vacuum, although vacuum drying can be used with caution. Plasmid DNA and other small circular DNAs can be vacuum-dried.
To help dissolve the DNA, carefully invert the tubes several times after adding buffer and tap the tube gently on the side. Alternatively let the DNA stand in buffer overnight at +2 to +8°C. Minimize vortexing of genomic DNA since this can cause shearing.
Avoid vigorous pipetting. Pipetting genomic DNA through narrow tip openings causes shearing or nicking. One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA. Regular pipette tips pose no problem for plasmid DNA and other small fragments.
|Determination of concentration, yield, and purity of DNA|
|DNA yields are determined from the concentration of DNA in the eluate, measured by absorbance at 260 nm. Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. Pure DNA has an A260/A280 ratio of 1.7–2.1. Absorbance readings at 260 nm should be between 0.1 and 1.0. Sample dilution should be adjusted accordingly. Use elution buffer or water (as appropriate) to dilute samples and to calibrate the spectrophotometer. Measure the absorbance at 260 and 280 nm, or scan absorbance from 220–320 nm (a scan will show if there are other factors affecting absorbance at 260 nm).|