Team:Lyon-INSA-ENS/Realisation/Protocols/Isolation

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<p style="text-align : center"> <font color="green" size="5"> Plasmid or Cosmid DNA Purification Using QIAGEN HiSpeed Plasmid Midi Kits </font> </p>
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<p style="text-align : center"> <font color="green" size="5"> NucleoSpin Plasmid QuickPure : Isolation of high-copy plasmid DNA from E. coli
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</font> </p>
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This protocol is for a preparation of up to 200µg of high- or low-copy plasmid or cosmid DNA using the QIAGEN HiSpeed Plasmid Midi Kit.
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This protocol is for a preparation of up to 15µg of high-copy plasmid or DNA using the Macherey-Nagel Nucleospin plasmid QuickPure Kit.
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A final DNA concentration of up to 0.4µg/µL can be expected, if eluting a high-copy plasmid with 500µL of Buffer TE.  
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<b>1.</b> Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5mL LB medium containing the appropriate selective antibiotic. Incubate for approx. 8h at 37°C with vigorous shaking (approx. 300 rpm).
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<b>1.</b>Use 1-5mL ( we used 1.5mL ) of a saturated E.Coli LB culture, pellet cells in a standard benchtop microcentrifuge for 30 s at
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11,000 x g. Discard the supernatant and remove as much of the liquid as possible.
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<b>2.</b> Dilute the starter 1/500 to 1/1000 into selective LB medium. For high-copy plasmids, inoculate 50mL medium. Grow at 37°C for 12-16h with vigorous shaking (approx. 300 rpm).  
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<b>2.</b> Add 250 μL Buffer A1. Resuspend the cell pellet
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completely by vortexing or pipetting up and down. Make
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sure no cell clumps remain before addition of Buffer A2!
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<b>3.</b> Harvest the bacterial cells by centrifugation at 6000 xg for 15 min at 4°C.
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<b>3.</b> Check Buffer A2 for precipitated SDS prior to
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use. If a white precipitate is visible, warm the buffer for
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several minutes at 30 – 40 °C until precipitate is dissolved
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completely. Cool buffer down to room temperature
 +
(18 – 25 °C).
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<b>4.</b> Resuspend the bacterial pellet in 6 mL Buffer P1.
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<b>4.</b> Add 250 μL Buffer A2. Mix gently by inverting the tube
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6 – 8 times. Do not vortex to avoid shearing of genomic
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DNA. Incubate at room temperature for up to 5 min or
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until lysate appears clear.
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<b>5.</b> Add 6 mL of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times, and incubate at room temperature (15-25°C) for 5 min. During the incubation prepare the QIAfilter Cartridge. Screw the cap onto the outlet nozzle of the QIAfilter Midi Cartridge. Place the filter into a convenient tube or a QIArack.
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<b>5.</b> Add 300 μL Buffer A3. Mix thoroughly by inverting the
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tube 6 – 8 times. Do not vortex to avoid shearing of
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genomic DNA!
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<b>6.</b> Add 6mL of chilled Buffer P3 to the lysate, and mix immediately and thoroughly by vigorously inverting 4-6 times. Do not incubate the lysate on ice.
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<b>6.</b> Centrifuge for 5 min at 11,000 x g at room temperature.
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Repeat this step in case the supernatant is not clear!
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<b>7.</b> Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for 10 min. Do not insert the plunger !
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<b>7.</b> Place a NucleoSpin Plasmid Column
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in a Collection Tube (2 mL) and decant the supernatant
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from step 3 or pipette a maximum of 750 μL of the
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supernatant onto the column. Centrifuge for 1 min
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at 11,000 x g. Discard flow-through and place the
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NucleoSpin Plasmid Column back into
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the collection tube.
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<b>8.</b> Equilibrate a HiSpeed Midi Tip by applying 4 mL Buffer QBT and allow the column to empty by gravity flow.
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<b>8.</b> Add 600 μL Buffer A4 (supplemented with ethanol ). Centrifuge for 1 min at 11,000 x g.
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Discard flow-through and place the NucleoSpin
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Plasmid Column back into the empty
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collection tube.
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<b>9.</b> Remove the cap from the QIAfilter outlet nozzle. Gently insert the plunger into the QIAfilter Midi Cartridge and filter the cell lysate into the previously equilibrated HiSpeed Tip.
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<b>9.</b> Centrifuge for 2 min at 11,000 x g and discard the
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collection tube.
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<b>10.</b> Allow the cleared lysate to enter the resin by gravity flow.
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<b>10.</b> Place the NucleoSpin® Plasmid / Plasmid (NoLid) Column
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in a 1.5 mL microcentrifuge tube (not provided) and
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add 50 μL Buffer AE. Incubate for 1 min at room
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temperature. Centrifuge for 1 min at 11,000 x g.
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<b>11.</b> Wash the HiSpeed Midi Tip with 20 mL of Buffer QC.
 
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<b>12.</b> Elute DNA with 5 mL of Buffer QF.
 
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<b>13.</b> Precipitate DNA by adding 3,5mL of room-temperature isopropanol to the eluted DNA. Mix and incubate at room-temperature during 5 min.
 
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<b>14.</b> During the incubation remove the plunger from a 20 mL syringe and attach the QIAprecipitator Midi Module onto the outlet nozzle.
 
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<b>15.</b> Place the QIAprecipitator over a waste bottle, transfer the eluate/isopropanol mixture into the 20
 
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mL syringe, and insert the plunger. Filter the eluate/ isopropanol mixture through the QIA precipitator using constant pressure.
 
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<b>16.</b> Remove the QIAprecipitator from the 20 mL syringe and pull out the plunger. Re-attach the QIAprecipitator and add 2mL 70% ethanol to the syringe. Wash the DNA by inserting the plunger and pressing the ethanol through the QIAprecipitaor using constant pressure.
 
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<b>17.</b> Remove the QIAprecipitator from the 20 mL syringe and pull out the plunger. Attach the QIAprecipitator to the 20 mL syringe again, insert the plunger, and dry the membrane by pressing air through the QIAprecipitaot quickly and forcefully. Repeat this step.
 
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<b>18.</b> Dry the outlet nozzle of the QIAprecipitator with absorbent paper to prevent ethanol carryover.
 
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<b>19.</b> Remove the plunger from a new 5 mL syringe and attach the QIAprecipitator onto the outlet nozzle. Hold the outlet of the QIAprecipitator over a 1,5 mL collection tube. Add 1 mL of Buffer TE to the 5 mL syringe. Insert the plunger and elute the DNA into the collection tube using constant pressure.
 
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<b>20.</b> Remove the QIAprecipitator from the 5 mL syringe, pull out the plunger and reattach the QIAprecipitator to the 5 mL syringe.
 
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<b>21.</b> Transfer the eluate from step 19 to the 5 mL syringe and eluate for a second time into the seme 1,5 mL tube.
 
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Determination of yield.
 
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Agarose gel analysis.
 
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<small> This protocol is extracted from "HiSpeed Plasmid Purification Handbook" by <B>QIAGEN </B>. </small>
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<small> This protocol is extracted from "Plasmid DNA purification user manual" by <B>Macherey-Nagel </B>. </small>
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Revision as of 08:47, 28 July 2011





NucleoSpin Plasmid QuickPure : Isolation of high-copy plasmid DNA from E. coli





This protocol is for a preparation of up to 15µg of high-copy plasmid or DNA using the Macherey-Nagel Nucleospin plasmid QuickPure Kit.





Procedure



1.Use 1-5mL ( we used 1.5mL ) of a saturated E.Coli LB culture, pellet cells in a standard benchtop microcentrifuge for 30 s at 11,000 x g. Discard the supernatant and remove as much of the liquid as possible.


2. Add 250 μL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down. Make sure no cell clumps remain before addition of Buffer A2!


3. Check Buffer A2 for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30 – 40 °C until precipitate is dissolved completely. Cool buffer down to room temperature (18 – 25 °C).


4. Add 250 μL Buffer A2. Mix gently by inverting the tube 6 – 8 times. Do not vortex to avoid shearing of genomic DNA. Incubate at room temperature for up to 5 min or until lysate appears clear.


5. Add 300 μL Buffer A3. Mix thoroughly by inverting the tube 6 – 8 times. Do not vortex to avoid shearing of genomic DNA!


6. Centrifuge for 5 min at 11,000 x g at room temperature. Repeat this step in case the supernatant is not clear!


7. Place a NucleoSpin Plasmid Column in a Collection Tube (2 mL) and decant the supernatant from step 3 or pipette a maximum of 750 μL of the supernatant onto the column. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin Plasmid Column back into the collection tube.


8. Add 600 μL Buffer A4 (supplemented with ethanol ). Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin Plasmid Column back into the empty collection tube.


9. Centrifuge for 2 min at 11,000 x g and discard the collection tube.


10. Place the NucleoSpin® Plasmid / Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube (not provided) and add 50 μL Buffer AE. Incubate for 1 min at room temperature. Centrifuge for 1 min at 11,000 x g.





This protocol is extracted from "Plasmid DNA purification user manual" by Macherey-Nagel .




ENS assystem Biomérieux INSA INSA
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