8.Move the QIAGEN column to a sawed-off 15-ml tube (in the QIAGEN materials box) and press the tube down over a new, labelled 1.5 ml microfuge tube so that the lid folds down over the edge of a microfuge tube rack.Elute DNA into the microfuge tube with 0.8 ml (800 ml) Buffer QF.
Flow begins when Buffer QF is added.Drain the QIAGEN-tip by allowing it to empty by gravity flow.
-Remove a 50 µl sample of the eluate and save for an analytical gel (sample 4).
9.Precipitate DNA with 0.7 volumes (560 ml, if you used 800 ml elution volume) of room-temperature isopropanol.Centrifuge immediately @ 21,000 rpm for 15 min in the Beckman Avanti 30 (F2402 rotor), and pour off the supernatant.
Precipitation of DNA with isopropanol should be carried out with all solution equilibrated to room temperature in order to minimize salt precipitation.Isopropanol pellets have a glassy appearance and may be more difficult to see than the flu salt-containing pellets that result from ethanol precipitation.It is a good idea to mark the outside of the tube before centrifugation, so that the pellet can be more easily located.
10.Add 1 ml of cold 70% ethanol to the tube with the pellet, being careful not to disturb the pellet.Centrifuge immediately @ 21,000 rpm for 10 min in the Beckman Avanti 30 (F2402 rotor).Dry for 2-3 min in the speed vac on low setting.Check after 2 min for smell of alcohol; if none, add 20 ml of sterile, distilled, deionized water.Otherwise dry a minute longer and check again.The pellet should not be completely dry -- a small amount of water will remain after all alcohol is gone.If you Speed-vac it to dryness, it will be very hard to redissolve.
The DNA pellet should be washed briefly in 70% ethanol, and then recentrifuge The 70% ethanol serves to remove precipitated salt, as well as to replace isopropanol with the more volatile ethanol, making the DNA easier to redissolve.A second was with room-temperature 70% ethanol may improve results in more sensitive application such as transfection and sequencing.After careful and complete removal of ethanol the pellet should be air-dried before redissolving in a appropriate volume of TE buffer (use high-quality water instead if it’s to be sequenced).Overdrying the pellet will make the DNA difficult to redissolve.Resuspend the DNA pellet by rinsing the walls to recover all the DNA. Pipetting the DNA up and down to promote resuspension may cause shearing, and should be avoided.The DNA may also be difficult to dissolve if it is too acidic.DNA dissolves best under slightly alkaline conditions.
11. To determine the yield, measure DNA concentration in a UV spectrophotometer.(followed by analysis on an agarose gel).Readings on a spectrophotometer are not always accurate, particularly if a single wavelength measurement is taken rather than a scan.
Analytical gel (optional): to analyze the purification procedure as shown in Figure 2(Appendix A, page 19), precipitate samples 1-4 (from steps 4-8) with 35 µ isopropanol.Rinse the pellets with 70% ethanol, drain well, air-dry, and resuspend 10 µl of TE, pH 8.0. Use 2 µi of each for analysis on a 1 % agarose gel (1).
