Team:Macquarie Australia/Protocols/Plasmid Prep

From 2013.igem.org

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<h1>Plasmid Prep</h1>
<h1>Plasmid Prep</h1>
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<b>QIAprep Spin Miniprep Kit Protocol</b>
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https://static.igem.org/mediawiki/2013/4/4f/PlasmidPrep2.jpg</center>
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<br><br>
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<b>1)</b> Pellet 1-5 mL bacterial culture by centrifugation at >8000 rpm for 3 minutes at room temperature.<br><br>
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<b>2)</b> Resuspend Pelleted bacterial cells in 250 uL Buffer P1.<br><br>
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<center><b>QIAprep Spin Miniprep Kit Protocol</b></center><br><br>
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<b>3)</b> Add 250 uL Buffer P2 and invert tube 4-6 times.<br><br>
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1) Pellet 1-5 mL bacterial culture by centrifugation at >8000 rpm for 3 minutes at room temperature.<br><br>
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<b>4)</b> Add 350 uL Buffer N3 and invert immediately 4-6 times and centrifuge for 10 minutes at 13,000 rpm.<br><br>
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2) Resuspend Pelleted bacterial cells in 250 uL Buffer P1.<br><br>
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<b>5)</b> Apply the supernatant to a QIAprep spin column and centrifuge for 30 seconds. Discard the flow-through.<br><br>
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3) Add 250 uL Buffer P2 and invert tube 4-6 times.<br><br>
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<b>6)</b> Wash the QIAprep spin column with 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.<br><br>
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4) Add 350 uL Buffer N3 and invert immediately 4-6 times and centrifuge for 10 minutes at 13,000 rpm.<br><br>
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<b>7)</b> Wash the QIAprep spin column with 0.75 mL Buffer PE and centrifuge for 30 seconds. Discard the flow-through.<br><br>
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5) Apply the supernatant to a QIAprep spin column and centrifuge for 30 seconds. Discard the flow-through.<br><br>
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<b>8)</b> Centrifuge for 1 minute to remove residual wash buffer.<br><br>
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6) Wash the QIAprep spin column with 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.<br><br>
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<b>9)</b> Place the QIAprep spin column into a clean Eppendorf. Elute DNA by adding 50 uL Buffer EB directly to the filter. Tap the column and stand for 1 minute and then centrifuge for 1 minute.<br><br>
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7) Wash the QIAprep spin column with 0.75 mL Buffer PE and centrifuge for 30 seconds. Discard the flow-through.<br><br>
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8) Centrifuge for 1 minute to remove residual wash buffer.<br><br>
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9) Place the QIAprep spin column into a clean Eppendorf. Elute DNA by adding 50 uL Buffer EB directly to the filter. Tap the column and stand for 1 minute and then centrifuge for 1 minute.<br><br>
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Latest revision as of 06:04, 13 September 2013


Plasmid Prep

QIAprep Spin Miniprep Kit Protocol

PlasmidPrep2.jpg



1) Pellet 1-5 mL bacterial culture by centrifugation at >8000 rpm for 3 minutes at room temperature.

2) Resuspend Pelleted bacterial cells in 250 uL Buffer P1.

3) Add 250 uL Buffer P2 and invert tube 4-6 times.

4) Add 350 uL Buffer N3 and invert immediately 4-6 times and centrifuge for 10 minutes at 13,000 rpm.

5) Apply the supernatant to a QIAprep spin column and centrifuge for 30 seconds. Discard the flow-through.

6) Wash the QIAprep spin column with 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.

7) Wash the QIAprep spin column with 0.75 mL Buffer PE and centrifuge for 30 seconds. Discard the flow-through.

8) Centrifuge for 1 minute to remove residual wash buffer.

9) Place the QIAprep spin column into a clean Eppendorf. Elute DNA by adding 50 uL Buffer EB directly to the filter. Tap the column and stand for 1 minute and then centrifuge for 1 minute.

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