CosmidDNAIsolation

實驗概要

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 10⁹ 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.]

實驗步驟

 1.Preparing Cell Lysate

   1)Harvest 3 ml cells by centrifuging at 9,000 x g for 15 minutes. Thoroughly remove all medium.

   2) Add 0.4 ml Resuspension Buffer (R3) containing 100 μg/ml RNase A  to the pellet and resuspend the cells until homogeneous. Transfer cell  suspension to a centrifuge tube.

   3)Add 0.4 ml Lysis Buffer (L7). Mix gently by inverting the capped  tube five times. Do not vortex. Incubate at room temperature for 5  minutes.

   4)Add 0.4 ml Precipitation Buffer (N3) and mix immediately by  inverting the tube until the mixture is homogeneous. Do not vortex.

   5)Centrifuge the mixture at >12,000 x g at room temperature for 10 minutes.

Note: If the pellet does not adhere to the bottom of the  tube, incubate the tube at room temperature for 5 minutes to allow the  separation of the lysate and gelatinous pellet. Pipette the clear lysate  into a sterile tube and centrifuge at >15,000 x g at room  temperature for 5 minutes to remove any remaining cellular debris.

2. Binding and Washing DNA

    1)      Pipette the supernatant from Step 5 onto the equilibrated  column. Allow the solution in the column to drain by gravity flow.

    2)Wash the column twice with 2.5 ml Wash Buffer (W8). Allow the  solution in the column to drain by gravity flow after each wash. Discard  the flow-through.

3. Eluting and Precipitating DNA

    1) Place a sterile centrifuge tube (elution tube) under the column.

    2)  Add 0.9 ml Elution Buffer (E4) to the column to elute DNA.  Allow the solution to drain by gravity flow. Do not force out any  remaining solution. The elution tube contains the purified DNA. Discard  the column.

    3)  Add 0.63 ml of isopropanol to the elution tube. Mix and place on ice for 10 minutes.

    4)  Centrifuge the mixture at >15,000 x g at 4°C for 20 minutes. Carefully remove and discard the supernatant.

 Note: It is highly recommended to use a swinging rotor.

    5)Resuspend the DNA pellet in 1 ml 70% ethanol.

    6)  Centrifuge at >12,000 x g at 4°C for 5 minutes. Carefully remove and discard the supernatant.

    7)  Air-dry the pellet for 10 minutes.

    8)  Resuspend the DNA pellet in 50 μl TE Buffer (TE).

Note: If a fixed-angle rotor was used for the centrifugation, the  pellet will be spread over the tube walls. Make sure to wash off the  tube walls when resuspending the pellet.

4. Extracted Results

This procedure allows purification of a ~45 kb cosmid DNA with yields of ~4 μg DNA per 3 ml culture.