|
|
| (16 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| | {{:Team:Evry/template_vo}} | | {{:Team:Evry/template_vo}} |
| | <html> | | <html> |
| - | | + | <a href='https://2013.igem.org/Team:Evry/ Protocols/ 01' title=' To English page'> <img src='https://static.igem.org/mediawiki/2013/ 7/ 78/ Anglais. gif'/></a> |
| - | <a href='https://2013.igem.org/Team:Evry/ Protocoles/ 03' title=' Vers la page française'> <img src='https://static.igem.org/mediawiki/2013/ b/ b9/ Francais. jpg'/></a> | + | |
| | | | |
| | <h1 align='center'> Purification de plasmide </h1> | | <h1 align='center'> Purification de plasmide </h1> |
| Line 8: |
Line 7: |
| | <i>Protocole provenant du manuel de purification de plasmide de Macherey-Nagel</i><br><br> | | <i>Protocole provenant du manuel de purification de plasmide de Macherey-Nagel</i><br><br> |
| | | | |
| - | 1. Récupération des bactéries
| |
| - | Répartir la culture de E.coli LB dans des tubes de 2 mL et centrifuger pendant 30s à 11 000 g. Enlever ensuite le maximum de surnageant possible.
| |
| - |
| |
| - | .Lyse cellulaire
| |
| - | Ajouter 250 μL de Buffer A1 (resuspension buffer). Resuspendre les cellules à l'aide d'un vortex ou d'une pipette.
| |
| - | A refaire avec MN !
| |
| - |
| |
| - | Pre-chill Buffer P3 at 4°C <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>
| |
| - | Use a tube or flask with a volume of at least 4 times the volume of the culture.<br><br>
| |
| - |
| |
| - | <b>2.Dilute the starter culture</b> 1/500 to 1/1000 into selective LB medium. For high-copy
| |
| - | 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>
| |
| - | Use a flask or vessel with a volume of at least 4 times the volume of the culture. The
| |
| - | culture should reach a cell density of approximately 3–4 x 109 cells per milliliter,
| |
| - | which typically corresponds to a pellet wet weight of approximately 3 g/liter
| |
| - | medium.<br><br>
| |
| - |
| |
| - | <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>
| |
| - | If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.<br><br>
| |
| - |
| |
| - | <b>4. Resuspend the bacterial pellet in 4 ml Buffer P1.</b><br>
| |
| - | For efficient lysis it is important to use a vessel that is large enough to allow complete
| |
| - | 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
| |
| - | cell clumps remain.<br><br>
| |
| - |
| |
| - | <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>
| |
| - | Do not vortex, as this will result in shearing of genomic DNA. The lysate should
| |
| - | appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After
| |
| - | use, the bottle containing Buffer P2 should be closed immediately to avoid
| |
| - | acidification from CO2 in the air.<br>
| |
| - | If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after
| |
| - | addition of Buffer P2. Mixing should result in a homogeneously colored suspension.<br>
| |
| - | If the suspension contains localized colorless regions or if brownish cell clumps are
| |
| - | still visible, continue mixing the solution until a homogeneously colored suspension
| |
| - | is achieved.<br><br>
| |
| - |
| |
| - | <b>6. Add 4 ml of chilled Buffer P3</b>, mix immediately and thoroughly by
| |
| - | vigorously inverting 4–6 times, and incubate on ice for 15 min.<br>
| |
| - | Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After
| |
| - | addition of Buffer P3, a fluffy white material forms and the lysate becomes less
| |
| - | viscous. The precipitated material contains genomic DNA, proteins, cell debris, and
| |
| - | KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulfate
| |
| - | precipitation. If the mixture still appears viscous, more mixing is required to
| |
| - | completely neutralize the solution.<br>
| |
| - | If LyseBlue reagent has been used, the suspension should be mixed until all trace of
| |
| - | blue has gone and the suspension is colorless. A homogeneous colorless suspension
| |
| - | indicates that the SDS has been effectively precipitated.<br><br>
| |
| - |
| |
| - | <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
| |
| - | DNA promptly.<br>
| |
| - | Before loading the centrifuge, the sample should be mixed again. Centrifugation
| |
| - | should be performed in non-glass tubes (e.g., polypropylene). After centrifugation
| |
| - | the supernatant should be clear.<br>
| |
| - | Note: Instead of centrifugation steps 7 and 8, the lysate can be efficiently cleared by
| |
| - | filtration using a QIAfilter Kits or Cartridges (see www.qiagen.com/products/
| |
| - | 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>1. Culture des bactéries</b><br> |
| - | supernatant containing plasmid DNA promptly.<br>
| + | Placer les cellules en culture dans du milieu LB durant la nuit. |
| - | This second centrifugation step should be carried out to avoid applying suspended
| + | <br><br> |
| - | 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>
| + | |
| | | | |
| - | <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>2. Récupération des bactéries</b><br> |
| - | Flow of buffer will begin automatically by reduction in surface tension due to the
| + | Répartir la culture de E.coli LB dans des tubes de 2 mL et centrifuger à 11 000 x g pendant 30 secondes. <br>Enlever ensuite le maximum de surnageant possible.<br><br> |
| - | presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain
| + | |
| - | completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop
| + | |
| - | when the meniscus reaches the upper frit in the column.<br><br>
| + | |
| | | | |
| - | <b>10. Apply the supernatant from step 8 to the QIAGEN-tip</b> and allow it to enter the resin | + | <b>3. Lyse cellulaire</b> <br> |
| - | by gravity flow.<br>
| + | Ajouter 250 μL de Buffer A1 (tampon de resuspension). Resuspendre les cellules à l'aide d'un vortex ou d'une pipette. |
| - | The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long
| + | <br> |
| - | and becomes cloudy due to further precipitation of protein, it must be centrifuged
| + | Ajouter 250 μL de Buffer A2 (tampon de lyse). Mélanger légèrement en retournant les tubes 6 à 8 fois. Laisser à température ambiante jusqu'à ce que le lysat apparaisse clair.<br> |
| - | again or filtered before loading to prevent clogging of the QIAGEN-tip.<br><br>
| + | Ajouter 300 μL de Buffer A3 (tampon de neutralisation). Mélanger doucement en retournant les tubes 6 à 8 fois.<br><br> |
| | | | |
| - | <b>11. Wash the QIAGEN-tip </b>with 2 x 10 ml Buffer QC.<br> | + | <b>4. Clarification du lysat</b><br> |
| - | Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is
| + | Centrifuger pendant à 11 000 x g pendant 5 minutes . Répéter cette opération tant que le surnageant n'est pas clair.<br><br> |
| - | sufficient to remove all contaminants in the majority of plasmid DNA preparations.
| + | |
| - | The second wash is especially necessary when large culture volumes or bacterial
| + | |
| - | strains producing large amounts of carbohydrates are used.<br><br>
| + | |
| | | | |
| - | <b>12. Elute DNA with 5 ml Buffer QF.</b><br> | + | <b>5. Liaison de l'ADN<br></b> |
| - | Collect the eluate in a 15 ml or 50 ml tube (not supplied). Use of polycarbonate
| + | Placer une colonne NucleoSpin Plasmid dans un Collection Tube de 2 mL et y verser le surnageant obtenu à l'étape précédente. Centrifuger à 11 000 x g pendant 1 minute. Jeter le filtrat et replacer la colonne dans le tube.<br><br> |
| - | centrifuge tubes is not recommended as polycarbonate is not resistant to the alcohol
| + | |
| - | used in subsequent steps.<br><br>
| + | |
| | | | |
| - | <b>13. Precipitate DNA</b> by adding 3.5 ml (0.7 volume) room-temperature | + | <b>6. Lavage de la membrane<br></b> |
| - | isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for
| + | Ajouter 600 μL de Buffer A4 (tampon de lavage) dans lequel de l'éthanol aura été mis précedemment. Centrifuger à 11 000 x g pendant 1 minute. Jeter le filtrat et placer la colonne dans un nouveau Collection tube.<br><br> |
| - | 30 min at 4°C. Carefully decant the supernatant.<br>
| + | |
| - | All solutions should be at room temperature in order to minimize salt precipitation,
| + | |
| - | 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 | + | <b>7. Séchage de la membrane<br></b> |
| - | 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
| + | Centrifuger à 11 000 x g pendant 2 minutes et jeter le Collection tube.<br><br> |
| - | disturbing the pellet.<br>
| + | |
| - | The 70% ethanol removes precipitated
| + | |
| - | salt and replaces isopropanol with the more volatile ethanol, making the DNA easier
| + | |
| - | to redissolve.<br><br>
| + | |
| | | | |
| - | <b>15. Air-dry the pellet</b> for 5–10 min, and redissolve the DNA in a suitable volume of buffer | + | <b>8. Elution de l'ADN<br></b> |
| - | (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).<br> | + | Placer la colonne dans un tube de 1,5 mL et ajouter 50 μL de Buffer AE (tampon d'élution). Laisser à température ambiante pendant une minute et centrifuger 11 000 x g pendant 1 minute.<br> |
| - | Redissolve the DNA pellet by rinsing the walls to recover all the DNA, especially if
| + | Mesurer la concentration au nanodrop puis stocker les tubes à -20°C<br> |
| - | 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>
| + | |
| | | | |
| | <script type='text/javascript'>writeFooter()</script> | | <script type='text/javascript'>writeFooter()</script> |
| | </html> | | </html> |
Répartir la culture de E.coli LB dans des tubes de 2 mL et centrifuger à 11 000 x g pendant 30 secondes.
Ajouter 250 μL de Buffer A1 (tampon de resuspension). Resuspendre les cellules à l'aide d'un vortex ou d'une pipette.
Ajouter 250 μL de Buffer A2 (tampon de lyse). Mélanger légèrement en retournant les tubes 6 à 8 fois. Laisser à température ambiante jusqu'à ce que le lysat apparaisse clair.
Ajouter 300 μL de Buffer A3 (tampon de neutralisation). Mélanger doucement en retournant les tubes 6 à 8 fois.
Centrifuger pendant à 11 000 x g pendant 5 minutes . Répéter cette opération tant que le surnageant n'est pas clair.
Placer une colonne NucleoSpin Plasmid dans un Collection Tube de 2 mL et y verser le surnageant obtenu à l'étape précédente. Centrifuger à 11 000 x g pendant 1 minute. Jeter le filtrat et replacer la colonne dans le tube.
Ajouter 600 μL de Buffer A4 (tampon de lavage) dans lequel de l'éthanol aura été mis précedemment. Centrifuger à 11 000 x g pendant 1 minute. Jeter le filtrat et placer la colonne dans un nouveau Collection tube.
Placer la colonne dans un tube de 1,5 mL et ajouter 50 μL de Buffer AE (tampon d'élution). Laisser à température ambiante pendant une minute et centrifuger 11 000 x g pendant 1 minute.
"
