Team:Oxford/protocols/MiniPrep: Plasmid Extraction

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<h1>Transforming Competent Cells</h1><BR><BR>
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<h1>QIAprep Plasmid DNA Purification</h1>
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<p>&#8617; Back to other <html><a href="https://2014.igem.org/Team:Oxford/protocols">protocols</a></html>.</p>
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<li>Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE.<BR><BR>
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<li>After growing cell cultures overnight in LB, resuspend pelleted cells in 250 μl Buffer P1 and transfer it into a microcentrifuge tube. Make sure that RNase A has been added to Buffer P1 before.<BR><BR>
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<li>Split the thawed cells into 2x 100µl aliquots. <BR><BR>
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<li>Add 250 μl of Buffer P2 and mix gently by inverting the tube 6 times. The cell suspension should turn blue. Try to carry on with the next step after less than 5 minutes to make sure that the lysis reaction doesn't proceed for too long.<BR><BR>
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<li>To each aliquot add 1µl of plasmid DNA.<BR><BR>
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<li>Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.<BR><BR>
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<li>Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter. <BR><BR>
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<li>Centrifuge for 10 minutes at 13000 rpm (or 17900 x g). Apply the supernatants to a spin colums<BR><BR>
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<li>During this incubation time prepare the antibiotic agar plates: <BR><BR>
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<li>Centrifuge again for 30-60 seconds and discard the flow-through.<BR><BR>
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<li>Wash the spin column by adding 500 μl of the PB buffer and centrifuge for 30-60s.<BR><BR>
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<li>Discard the flow-through and wash the spin column again by adding 750 μl of PE buffer. Centrifuge for 1 minute.<BR><BR>
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<li>Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing.<BR><BR>
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<li>Discard the flow-through and centrifuge again for 1 minute. Now place the column in a 1.5ml microcentrifuge tube.<BR><BR>
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<li>Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment.<BR><BR>
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<li>Add 50 μl of the EB buffer to the centre of the spin column. Let stand for 2 minutes and centrifuge for 1 minute.<BR><BR>
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<li>Once cool take the agar and petri dishes to the laminar flow hood.<BR><BR>
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<li>Add the appropriate volume of antibiotic to the agar bottle before pouring.<BR><BR>
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<li>Pour the agar evenly to no higher than the raised line. NOTE: place the lid resting on the edge of the dish so as to catch any drops that fall from the bottle.<BR><BR>
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<li>Leave the agar plates to set (~15-30 minutes)<BR><BR>
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<li>Heat shock the bacteria by placing them in the 42ºC water bath for NO MORE THAN 45 SECONDS. <BR><BR>
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<li>Immediately place the bacteria on ICE for 1 minute. <BR><BR>
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<li>Add 800µl LB and incubate at 37ºC for 1 hour. <BR><BR>
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<li>Plate the bacteria under sterile conditions (bunsen burner ON): <BR><BR>
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<li>Place a glass spreader in ethanol. <BR><BR>
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<li>Flame the spreader to burn off the ethanol and let it cool. <BR><BR>
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<li>Plate #1: spread 100µl of cell culture onto the plate<BR><BR>
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<li>Plate #2: spin down the remaining cells from step 3. Discard the supernatant and resuspend the pellet in 100µl of fresh culture. Spread this onto a second plate as in previous step<BR><BR>
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<li>Incubate at 37ºC overnight. </li><BR><BR>
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Latest revision as of 15:11, 27 July 2014

QIAprep Plasmid DNA Purification

↩ Back to other protocols.

  1. After growing cell cultures overnight in LB, resuspend pelleted cells in 250 μl Buffer P1 and transfer it into a microcentrifuge tube. Make sure that RNase A has been added to Buffer P1 before.

  2. Add 250 μl of Buffer P2 and mix gently by inverting the tube 6 times. The cell suspension should turn blue. Try to carry on with the next step after less than 5 minutes to make sure that the lysis reaction doesn't proceed for too long.

  3. Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.

  4. Centrifuge for 10 minutes at 13000 rpm (or 17900 x g). Apply the supernatants to a spin colums

  5. Centrifuge again for 30-60 seconds and discard the flow-through.

  6. Wash the spin column by adding 500 μl of the PB buffer and centrifuge for 30-60s.

  7. Discard the flow-through and wash the spin column again by adding 750 μl of PE buffer. Centrifuge for 1 minute.

  8. Discard the flow-through and centrifuge again for 1 minute. Now place the column in a 1.5ml microcentrifuge tube.

  9. Add 50 μl of the EB buffer to the centre of the spin column. Let stand for 2 minutes and centrifuge for 1 minute.

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