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From 2013.igem.org

Purification de plasmide

Protocole provenant du manuel de purification de plasmide de Macherey-Nagel

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 (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 produit de lyse apparaisse clair.

Ajouter 300 μL de Buffer A3 (tampon de neutralisation). Mélanger doucement en retournant les tubes 6 à 8 fois.



. Clarification du produit de lyse Centrifuger pendant 5min à Pre-chill Buffer P3 at 4°C

1.Pick a single colony 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).
Use a tube or flask with a volume of at least 4 times the volume of the culture.

2.Dilute the starter culture 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).
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.

3. Harvest the bacterial cells 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.
If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.

4. Resuspend the bacterial pellet in 4 ml Buffer P1.
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.

5. Add 4 ml Buffer P2, mix thoroughly by vigorously inverting the sealedtube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.
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.
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.
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.

6. Add 4 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 15 min.
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.
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.

7. Centrifuge 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.
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.
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).

8. Centrifuge the supernatant again 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 supernatant containing plasmid DNA promptly.
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.

9. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
Flow of buffer will begin automatically by reduction in surface tension due to the 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.

10. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
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.

11. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC.
Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is 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.

12. Elute DNA with 5 ml Buffer QF.
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.

13. Precipitate DNA 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 30 min at 4°C. Carefully decant the supernatant.
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.
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.

14. Wash DNA pellet 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.
The 70% ethanol removes precipitated salt and replaces isopropanol with the more volatile ethanol, making the DNA easier to redissolve.

15. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).
Redissolve the DNA pellet by rinsing the walls to recover all the DNA, especially if 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.