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

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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">  
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Plasmid DNA purification using QIAprep® Spin Miniprep Kit </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 designed for purification of up to 20 µg of high-copy plasmid DNA from 1-5mL overnight cultures in LB mediul.
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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> Pellet 1-5mL bacterial overnight culture by centrifugation at >8000 rpm ( we used 13000 ) for 3mn at room temperature
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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> Resuspend pelleted bacterial cells in 250µL buffer P1 and transfer to a microcentrifuge tube
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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> Add 250µL buffer P2 and mix thoroughly by inverting the tybe 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5mn.
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<b>4.</b> Resuspend the bacterial pellet in 6 mL Buffer P1.
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<b>4.</b> Add 350µL buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
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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> Centrifuge for 10mn at 13000 rpm in a table-top microcentrifuge
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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> Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.
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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.Recommended if the strain is endA+</b> Wash the QIAprep spin column by adding 0.5mL buffer PB. Centrifuge for 30-60s and discard the flow-through.
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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> Wash the QIAprep spin column by adding 0.75 mL buffer PE. Centrifuge for 30-60s and discard the flow-through.
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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 1mn to remove residual wash buffer
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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 QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50 µL buffer EB ( 10mM Tris.Cl, pH 8.5 ), let stand for 1mn and centrifuge for 1mn.
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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 "QIAprep Miniprep Handbook" by <B>QIAGEN </B>. </small>
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Latest revision as of 10:44, 10 January 2012





Plasmid DNA purification using QIAprep® Spin Miniprep Kit





This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1-5mL overnight cultures in LB mediul.





Procedure



1. Pellet 1-5mL bacterial overnight culture by centrifugation at >8000 rpm ( we used 13000 ) for 3mn at room temperature


2. Resuspend pelleted bacterial cells in 250µL buffer P1 and transfer to a microcentrifuge tube


3. Add 250µL buffer P2 and mix thoroughly by inverting the tybe 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5mn.


4. Add 350µL buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.


5. Centrifuge for 10mn at 13000 rpm in a table-top microcentrifuge


6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.


7.Recommended if the strain is endA+ Wash the QIAprep spin column by adding 0.5mL buffer PB. Centrifuge for 30-60s and discard the flow-through.


8. Wash the QIAprep spin column by adding 0.75 mL buffer PE. Centrifuge for 30-60s and discard the flow-through.


9. Centrifuge for 1mn to remove residual wash buffer


10. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50 µL buffer EB ( 10mM Tris.Cl, pH 8.5 ), let stand for 1mn and centrifuge for 1mn.





This protocol is extracted from "QIAprep Miniprep Handbook" by QIAGEN .




ENS assystem Biomérieux INSA INSA
Retrieved from "http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Miniprep"