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| - | <h1 align='center'> Protocol for Plasmid purification </h1> | + | <h1 align='center'> Plasmid purification </h1> |
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| - | <i>Protocol from Macherey-Nagel plasmid purification handbook</i><br><br> | + | <i>Protocole from Macherey-Nagel plasmid purification notebook</i><br><br> |
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| - | A refaire avec MN !
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| - | Pre-chill Buffer P3 at 4°C <br><br>
| + | <b>1. Cell culture</b><br><br> |
| - | <b>1.Pick a single colony</b> from a freshly streaked selective plate and inoculate a starter | + | |
| - | culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate
| + | |
| - | for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).<br>
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| - | Use a tube or flask with a volume of at least 4 times the volume of the culture.<br><br>
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| | | | |
| - | <b>2.Dilute the starter culture</b> 1/500 to 1/1000 into selective LB medium. For high-copy
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| - | plasmids, inoculate 25 ml medium with 25–50 μl of starter culture. For low-copy plasmids, inoculate 100 ml medium with 100–200 μl of starter culture. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).<br>
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| - | Use a flask or vessel with a volume of at least 4 times the volume of the culture. The
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| - | culture should reach a cell density of approximately 3–4 x 109 cells per milliliter,
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| - | which typically corresponds to a pellet wet weight of approximately 3 g/liter
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| - | medium.<br><br>
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| - | <b>3. Harvest the bacterial cells</b> by centrifugation at 6000 x g (6900rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo) for 15 min at 4°C.<br> | + | <b>2. Cell harvesting</b><br> |
| - | If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.<br><br>
| + | Set saturated E.coli LM culture into 2 mL tubes. Centrifuge at 11 000 x g for 30 secondes. Discard as much as supernatant as possible.<br><br> |
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| - | <b>4. Resuspend the bacterial pellet in 4 ml Buffer P1.</b><br> | + | <b>3. Cell lysis</b> <br> |
| - | For efficient lysis it is important to use a vessel that is large enough to allow complete
| + | Add 250 μL Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette. |
| - | mixing of the lysis buffers. Ensure that RNase A has been added to Buffer P1.If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure LyseBlue particles are completely resuspended. The bacteria should be resuspended completely by vortexing or pipetting up and down until no
| + | <br> |
| - | cell clumps remain.<br><br>
| + | Add 250 μL Buffer A2 (lysis buffer). Mix gently by inverting the tube 6 - 8 times. Incubate at room temperature until lysate appears clear.<br> |
| | + | Add 300 μL Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .<br><br> |
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| - | <b>5. Add 4 ml Buffer P2</b>, mix thoroughly by vigorously inverting the sealedtube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.<br> | + | <b>4. Lysate clarification</b><br> |
| - | Do not vortex, as this will result in shearing of genomic DNA. The lysate should
| + | Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.<br><br> |
| - | appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After
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| - | use, the bottle containing Buffer P2 should be closed immediately to avoid
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| - | acidification from CO2 in the air.<br>
| + | |
| - | If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after
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| - | addition of Buffer P2. Mixing should result in a homogeneously colored suspension.<br>
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| - | If the suspension contains localized colorless regions or if brownish cell clumps are
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| - | still visible, continue mixing the solution until a homogeneously colored suspension
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| - | is achieved.<br><br> | + | |
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| - | <b>6. Add 4 ml of chilled Buffer P3</b>, mix immediately and thoroughly by | + | <b>5. DNA Binding<br></b> |
| - | vigorously inverting 4–6 times, and incubate on ice for 15 min.<br>
| + | Place a NucleoSpin Plasmid Column in a Collection Tube of 2 mL et and set the supernatant from the last step. Centrifuge at 11 000 x g for 1 minute. Discard flow-through and place the column back into the collection tube.<br><br> |
| - | Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After
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| - | addition of Buffer P3, a fluffy white material forms and the lysate becomes less
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| - | viscous. The precipitated material contains genomic DNA, proteins, cell debris, and
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| - | KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulfate
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| - | precipitation. If the mixture still appears viscous, more mixing is required to
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| - | completely neutralize the solution.<br>
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| - | If LyseBlue reagent has been used, the suspension should be mixed until all trace of
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| - | blue has gone and the suspension is colorless. A homogeneous colorless suspension
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| - | indicates that the SDS has been effectively precipitated.<br><br>
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| - | <b>7. Centrifuge</b> at ≥20,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 30 min at 4°C. Remove supernatant containing plasmid | + | <b>6. Membrane washing<br></b> |
| - | DNA promptly.<br>
| + | Add 600 μL Buffer A4 (wash buffer) previously supplemented with ethanol. Centrifuge ar 11 000 x g for 1 minute. Discard flow-through and place the column back into an empty collection tube.<br> |
| - | Before loading the centrifuge, the sample should be mixed again. Centrifugation
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| - | should be performed in non-glass tubes (e.g., polypropylene). After centrifugation
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| - | the supernatant should be clear.<br>
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| - | Note: Instead of centrifugation steps 7 and 8, the lysate can be efficiently cleared by
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| - | filtration using a QIAfilter Kits or Cartridges (see www.qiagen.com/products/
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| - | plasmid/LargeScaleKits).<br><br>
| + | |
| | | | |
| - | <b>8. Centrifuge the supernatant again</b> at ≥20,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo) for 15 min at 4°C. Remove | + | <b>7. Dry membrane<br></b> |
| - | supernatant containing plasmid DNA promptly.<br>
| + | Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.<br><br> |
| - | This second centrifugation step should be carried out to avoid applying suspended
| + | |
| - | or particulate material to the QIAGEN-tip. Suspended material (causing the sample
| + | |
| - | to appear turbid) can clog the QIAGEN-tip and reduce or eliminate gravity flow.<br><br>
| + | |
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| - | <b> 9. Equilibrate a QIAGEN-tip 100 </b>by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.<br> | + | <b>8. DNA Elution<br></b> |
| - | Flow of buffer will begin automatically by reduction in surface tension due to the
| + | Place the column in a 1,5 mL and add 50 μL de Buffer AE (elution buffer). Incubate at room temperature and centrifuge at 11 000 x g for 1 minute.<br> |
| - | presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain
| + | Measure the concentration with nanodrop, then stock the tubes at -20°C<br> |
| - | completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop
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| - | when the meniscus reaches the upper frit in the column.<br><br>
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| - | | + | |
| - | <b>10. Apply the supernatant from step 8 to the QIAGEN-tip</b> and allow it to enter the resin
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| - | by gravity flow.<br>
| + | |
| - | The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long
| + | |
| - | and becomes cloudy due to further precipitation of protein, it must be centrifuged
| + | |
| - | again or filtered before loading to prevent clogging of the QIAGEN-tip.<br><br>
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| - | | + | |
| - | <b>11. Wash the QIAGEN-tip </b>with 2 x 10 ml Buffer QC.<br>
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| - | Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is
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| - | sufficient to remove all contaminants in the majority of plasmid DNA preparations.
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| - | The second wash is especially necessary when large culture volumes or bacterial
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| - | strains producing large amounts of carbohydrates are used.<br><br>
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| - | | + | |
| - | <b>12. Elute DNA with 5 ml Buffer QF.</b><br>
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| - | Collect the eluate in a 15 ml or 50 ml tube (not supplied). Use of polycarbonate
| + | |
| - | centrifuge tubes is not recommended as polycarbonate is not resistant to the alcohol
| + | |
| - | used in subsequent steps.<br><br>
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| - | | + | |
| - | <b>13. Precipitate DNA</b> by adding 3.5 ml (0.7 volume) room-temperature
| + | |
| - | isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for
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| - | 30 min at 4°C. Carefully decant the supernatant.<br>
| + | |
| - | All solutions should be at room temperature in order to minimize salt precipitation,
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| - | although centrifugation is carried out at 4°C to prevent overheating of the sample.<br>
| + | |
| - | Alternatively, disposable conical bottom centrifuge tubes can be used for
| + | |
| - | centrifugation at 5000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 60 min at 4°C. Isopropanol pellets have a glassy
| + | |
| - | appearance and may be more difficult to see than the fluffy, salt-containing pellets
| + | |
| - | that result from ethanol precipitation. Marking the outside of the tube before
| + | |
| - | centrifugation allows the pellet to be more easily located. Isopropanol pellets are also
| + | |
| - | more loosely attached to the side of the tube, and care should be taken when
| + | |
| - | removing the supernatant.<br><br>
| + | |
| - | | + | |
| - | <b>14. Wash DNA pellet</b> with 2 ml of room-temperature 70% ethanol, and
| + | |
| - | centrifuge at ≥15,000 x g (10000rpm with rotor AV10/Ser N.0106/04/Cat N.11175754 - Centri CR31 Thermo)for 10 min. Carefully decant the supernatant without
| + | |
| - | disturbing the pellet.<br>
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| - | The 70% ethanol removes precipitated
| + | |
| - | salt and replaces isopropanol with the more volatile ethanol, making the DNA easier
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| - | to redissolve.<br><br>
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| - | | + | |
| - | <b>15. Air-dry the pellet</b> for 5–10 min, and redissolve the DNA in a suitable volume of buffer
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| - | (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).<br>
| + | |
| - | Redissolve the DNA pellet by rinsing the walls to recover all the DNA, especially if
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| - | glass tubes have been used. Pipetting the DNA up and down to promote
| + | |
| - | resuspension may cause shearing and should be avoided. Overdrying the pellet will
| + | |
| - | make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline
| + | |
| - | conditions; it does not easily dissolve in acidic buffers.<br><br>
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Set saturated E.coli LM culture into 2 mL tubes. Centrifuge at 11 000 x g for 30 secondes. Discard as much as supernatant as possible.
Add 250 μL Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette.
Add 250 μL Buffer A2 (lysis buffer). Mix gently by inverting the tube 6 - 8 times. Incubate at room temperature until lysate appears clear.
Add 300 μL Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .
Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.
Place a NucleoSpin Plasmid Column in a Collection Tube of 2 mL et and set the supernatant from the last step. Centrifuge at 11 000 x g for 1 minute. Discard flow-through and place the column back into the collection tube.
Add 600 μL Buffer A4 (wash buffer) previously supplemented with ethanol. Centrifuge ar 11 000 x g for 1 minute. Discard flow-through and place the column back into an empty collection tube.
Place the column in a 1,5 mL and add 50 μL de Buffer AE (elution buffer). Incubate at room temperature and centrifuge at 11 000 x g for 1 minute.
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