MaxipreppreparationofPlasmidDNA

實驗概要

The  PureLink? HiPure Plasmid DNA Maxiprep Kit allows purification of  500–850 μg of high-quality plasmid DNA from 100–200 mL overnight E. coli cultures in ~2 hours when cloning high copy number plasmids.

實驗原理

The  PureLink? HiPure Plasmid Filter Purification Kits allow isolation of  high yields of highly pure plasmid DNA. The HiPure Filter Column  provides rapid clearing of the bacterial lysate without the need for  centrifugation. The lysate Filtration Cartridge is integrated into the  DNA Binding Column and combines the steps of clearing the bacterial  lysate with binding the DNA directly to the anionexchange resin. The  HiPure Filter Column protocol reduces time and effort for plasmid  purification. 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  plasmid DNA purity is equivalent to two passes through CsCl gradients  and has low endotoxin levels.

主要試劑

Resuspension Buffer (R3) with RNase A

Lysis Buffer (L7)

Precipitation Buffer (N3)

Equilibration Buffer (EQ1)

Wash Buffer (W8)

Elution Buffer (E4)

TE Buffer (TE)

HiPure Filter Maxi Columns

Column Holder

PureLink? HiPure Precipitator Module (supplied with cat. nos. K2100-26 and K2100-27 only)

Note: For  Maxipreps of low copy number plasmids from bacterial cultures of  >200 mL, use twice the amount of Resuspension Buffer (R3), Lysis  Buffer (L7), and Precipitation Buffer (N3) as directed in the protocol.

Order  the PureLink? HiPure BAC Buffer kit for additional buffers if the  buffers in the kit are insufficient for using all of the columns when  following this protocol.

實驗材料

Overnight culture of transformed E. coli cells

Isopropanol

70% ethanol

Sterile, microcentrifuge tubes

PureLink? Nucleic Acid Purification Rack

Tubes or centrifuge bottles for harvesting cells

Centrifuge and rotor appropriate for harvesting cells

50-mL centrifuge tubes capable of withstanding centrifugation forces >12,000 × g

Centrifuge capable of centrifuging at >12,000 × g at 4°C

實驗步驟

1.        Before Starting

Verify that the Resuspension Buffer (R3) contains RNase A, and no precipitate has formed in the Lysis Buffer (L7).

2.        Equilibrating the Column

The PureLink? HiPure Filter Maxi Columns are prepackaged with the Filtration cartridge inserted into the column housing.

1)      Use  the Column Holder to support a HiPure Filter Maxi Column in the mouth a  flask, or place the Maxi Column on the PureLink? Nucleic Acid  Purification Rack (see manual supplied with the rack for more details).

2)      Apply 30 mL Equilibration Buffer (EQ1) directly into the Filtration Cartridge, which is inserted into the Maxi Column.

3)      Allow the solution in the HiPure Filter Maxi Column to drain by gravity flow.

4)      Prepare the cell lysate (see below) while the HiPure Filter Maxi Column is equilibrating.

3.        Preparing Cell Lysate

1)      For  high copy number plasmids, use 100–200 mL of an overnight LB culture  per sample. For low copy number plasmids, harvest 250–500 mL of an  overnight LB culture per sample.

2)      Harvest the cells by centrifuging the overnight LB culture at 4,000 × g for 10 minutes. Remove all medium.

3)      Add 10 mL Resuspension Buffer (R3) with RNase A to the pellet and resuspend the cells until homogeneous.

4)      Add  10 mL Lysis Buffer (L7). Mix gently by inverting the capped tube until  the lysate mixture is thoroughly homogeneous. Do not vortex. Incubate at  room temperature for 5 minutes. Note: Do not allow lysis to proceed for  more than 5 minutes.

5)      Add  10 mL Precipitation Buffer (N3) and mix immediately by inverting the  tube until the mixture is thoroughly homogeneous. Do not vortex.

6)      Proceed to Loading Filter Column and Washing DNA

4.        Loading Filter Column and Washing DNA

1)      Transfer  the precipitated lysate from Step 5 in Preparing Cell Lysate including  all the precipitated material into the equilibrated HiPure Filter Maxi  Column. Let the lysate run through the filter by gravity flow until the  flow stops (10–15 minutes) or becomes very slow (<1 drop per 10  seconds). Discard the flow through.

2)      Optional:  The final DNA yield may be increased by washing the residual bacterial  lysate in the HiPure Filter Maxi Column with 10 mL Wash Buffer (W8).  Again, let the buffer flow through the HiPure Filter Maxi Column by  gravity flow until the flow stops or dripping becomes very slow.

3)      Immediately  after the HiPure Filter Maxi Column has stopped dripping, remove the  inner Filtration Cartridge from the column and discard. Note: Use the  HiPure Filtration Cartridge only once. The cartridge is for single use  only.

4)      Wash  the Maxi column with 50 mL of Wash Buffer (W8). Allow the solution in  the column to drain by gravity flow. Discard the flow-through.

5)      Proceed to Eluting DNA, (below).

5.        Eluting DNA

1)      Place a sterile 50-mL centrifuge tube (elution tube) under the HiPure Filter Maxi column.

2)      Add  15 mL Elution Buffer (E4) to the Maxi column to elute the DNA. Allow  the solution to drain by gravity flow. Do not force out any remaining  solution. The elution tube contains the purified DNA.

3)      Discard the HiPure Filter Maxi column.

4)      Proceed to Precipitating DNA with Isopropanol, (below) or Precipitating DNA Using Precipitator Module

6.        Precipitating DNA with Isopropanol

1)      Add 10.5 mL isopropanol to the DNA to the elution tube. Mix well.

2)      Centrifuge the tube at >12,000 × g for 30 minutes at 4°C. Carefully remove and discard the supernatant.

3)      Add 5 mL 70% ethanol to resuspend the DNA pellet.

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

5)      Air-dry the pellet for ~10 minutes.

6)      Resuspend the DNA pellet in 500 μL TE Buffer (TE). For low copy number plasmids, use 200 μL TE Buffer (TE).

7.        Storing DNA

Store the purified DNA at –20°C, or proceed to desired downstream application.

注意事項

1.        DNA  precipitation can be completed using centrifugation or with the  PureLink? HiPure Precipitator Module (included with the FP Maxiprep Kit  or purchased as a separate kit). The precipitator module allows DNA  precipitation within 10 minutes without any centrifugation steps. Refer  to the section below to precipitate DNA with isopropanol by  centrifugation. For DNA precipitation using the PureLink? HiPure  Precipitator Module. For a detailed protocol on using the precipitator  module, refer to the product insert included with the precipitator.

2.        Occasionally,  insoluble particles may be present. These particles do not influence  the quality of the DNA and can be easily removed. To remove insoluble  particles, centrifuge the DNA solution at high speed at room temperature  for 1 minute. Transfer the supernatant (DNA sample) into a fresh tube.

3.        To  avoid repeated freezing and thawing of DNA, store the purified DNA at  4°C for immediate use or aliquot the DNA and store at –20°C for  long-term storage.