Isolation of high yields of highly pure cosmid DNA using PureLink? HiPure Plasmid Purification Kits. ?

實驗原理 The PureLink? HiPure Plasmid Purification Kits allow isolation of high yields of highly pure cosmid DNA. The kits are designed to efficiently isolate plasmid DNA from E. coli in 1.5-2.5 hours using anion-exchange columns without the use of any organic solvents or cesium chloride (CsCl). The isolated cosmid DNA is of high purity, equivalent to two passes through CsCl gradients, and contains low endotoxin levels. The PureLink? HiPure Plasmid DNA Purification Kits are available in three formats that allow you to purify cosmid DNA using different starting culture volumes. ?

主要試劑 Resuspension Buffer (R3)[details：Add RNase A to the Resuspension Buffer (R3) according to instructions on the label of the bottle. Mix well. Mark the bottle label to indicate that RNase A is added. Store the buffer with RNase at 4° C. ] Lysis Buffer (L7)[details：Check the Lysis Buffer (L7) for precipitates. If present, warm the solution briefly at 37° C to dissolve the precipitate. Verify that no precipitate has formed in the Lysis Buffer (L7). ] Equilibrating the Column [details：Place the PureLink? HiPure column on the PureLink? Nucleic Acid Purification Rack. Apply Equilibration Buffer (EQ1) to the column. Allow the solution in the column to drain by gravity flow. ? ?

實驗材料 Bacterial Cultures [details：Grow transformed E. coli in LB medium with the appropriate antibiotic. The bacterial culture should have a cell density of approximately 10 9 ?cells/ml or an absorbance at 600 nm (A600) of 1-1.5. Use bacterial culture in transition between exponential phase and stationary phase.] Bacterial Cultures For Cosmid [details：Inoculate a bacterial culture containing your cosmid construct in medium with the appropriate selective antibiotic and grow the bacteria for 16 h (or overnight) with 225 rpm shaking. Add 20 mg/ml RNase A to Resuspension Buffer (R3) to a final concentration of 100 μg/ml.] ?