Team:CSU Fort Collins/Notebook/Protocols=Miniprep
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<ul> | <ul> | ||
<li><a href='/Team:CSU_Fort_Collins/Members/'><span>Members</span></a></li> | <li><a href='/Team:CSU_Fort_Collins/Members/'><span>Members</span></a></li> | ||
- | <li class='last'><a href='/Team:CSU_Fort_Collins/ | + | <li><a href='/Team:CSU_Fort_Collins/Sponsors/'><span>Sponsors</span></a></li> |
+ | <li class='last'><a href='/Team:CSU_Fort_Collins/Acknowledgements/'><span>Acknowledgements</span></a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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<li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/DailyNotes'><span>Daily Notes</span></a> | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/DailyNotes'><span>Daily Notes</span></a> | ||
<ul> | <ul> | ||
- | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Biosensor/'><span>Biosensor</span></a> | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Biosensor/Jun'><span>Biosensor</span></a> |
<ul> | <ul> | ||
- | <li><a href=" | + | <li><a href="/Team:CSU_Fort_Collins/Notebook/Biosensor/Jun"><span>June</span></a></li> |
- | <li><a href=" | + | <li><a href="/Team:CSU_Fort_Collins/Notebook/Biosensor/Jul"><span>July</span></a></li> |
- | + | <li class='last'><a href='/Team:CSU_Fort_Collins/Notebook/Biosensor/Aug'><span>August</span></a></li> | |
- | <li class='last'><a href= | + | |
</ul> | </ul> | ||
</li> | </li> | ||
- | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/'><span>Breakdown</span></a> | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul'><span>Breakdown</span></a> |
<ul> | <ul> | ||
- | <li><a href=" | + | <li><a href="/Team:CSU_Fort_Collins/Notebook/Breakdown/Jul"><span>July</span></a></li> |
- | <li><a href=' | + | <li><a href='/Team:CSU_Fort_Collins/Notebook/Breakdown/Aug'><span>August</span></a></li> |
- | <li class='last'><a href=" | + | <li class='last'><a href="/Team:CSU_Fort_Collins/Notebook/Breakdown/Sep"><span>September</span></a></li> |
</ul> | </ul> | ||
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- | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/HVP/'><span>High-Value Product</span></a> | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Notebook/HVP/Jun'><span>High-Value Product</span></a> |
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- | + | ||
- | <li class='last'><a href=" | + | |
</ul> | </ul> | ||
</li> | </li> | ||
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- | <li class='last'><a href=" | + | |
</ul> | </ul> | ||
</li> | </li> | ||
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<ul> | <ul> | ||
<li><a href='/Team:CSU_Fort_Collins/Collab/'><span>Collaboration</span></a></li> | <li><a href='/Team:CSU_Fort_Collins/Collab/'><span>Collaboration</span></a></li> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
- | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/ | + | <li class='has-sub'><a href='/Team:CSU_Fort_Collins/Parts/'><span>Achievements</span></a> |
<ul> | <ul> | ||
<li><a href='/Team:CSU_Fort_Collins/Parts/'><span>Parts</span></a></li> | <li><a href='/Team:CSU_Fort_Collins/Parts/'><span>Parts</span></a></li> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
- | <li><a href='/Team:CSU_Fort_Collins/Safety/'><span>Safety</span></a></li> | + | <li class='last'><a href='/Team:CSU_Fort_Collins/Safety/'><span>Safety</span></a></li> |
</ul> | </ul> | ||
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<p> | <p> | ||
- | |||
Before starting: | Before starting: | ||
<ul> | <ul> | ||
<li>Add RNase A to Resuspension Buffer (R3) according to the instructions on the label; mix well</li> | <li>Add RNase A to Resuspension Buffer (R3) according to the instructions on the label; mix well</li> | ||
- | <li>Mark on the label that RNase A is added. Store Buffer R3 with RNase A at 4 | + | <li>Mark on the label that RNase A is added. Store Buffer R3 with RNase A at 4 °C</li> |
- | <li>Warm Lysis Buffer (L7) briefly at 37 | + | <li>Warm Lysis Buffer (L7) briefly at 37 °C to redissolve any particulate matter</li> |
<li>Add 100% ethanol to Wash Buffer (W9) and Wash Buffer (W10) according to the instructions on each label; mix well</li> | <li>Add 100% ethanol to Wash Buffer (W9) and Wash Buffer (W10) according to the instructions on each label; mix well</li> | ||
<li>Store wash buffers with ethanol at room temperature</li> | <li>Store wash buffers with ethanol at room temperature</li> | ||
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<li>Resuspend</li> | <li>Resuspend</li> | ||
<ul> | <ul> | ||
- | <li>Add 250 | + | <li>Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet</li> |
<li>Resuspend pellet until it is homogeneous</li> | <li>Resuspend pellet until it is homogeneous</li> | ||
</ul> | </ul> | ||
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Note: This step is time sensitive. | Note: This step is time sensitive. | ||
<ul> | <ul> | ||
- | <li>Add 250 | + | <li>Add 250 μL Lysis Buffer (L7)</li> |
<li>Mix gently by inverting the capped tube five times (Do NOT vortex)</li> | <li>Mix gently by inverting the capped tube five times (Do NOT vortex)</li> | ||
<li>Incubate the tube at room temperature for 5 minutes</li> | <li>Incubate the tube at room temperature for 5 minutes</li> | ||
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<li>Precipitate</li> | <li>Precipitate</li> | ||
<ul> | <ul> | ||
- | <li>Add 350 | + | <li>Add 350 μL Precipitation Buffer (N4)</li> |
<li>Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous (Do NOT vortex)</li> | <li>Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous (Do NOT vortex)</li> | ||
<li>Centrifuge the lysate at >12,000 X g for 10 minutes</li> | <li>Centrifuge the lysate at >12,000 X g for 10 minutes</li> | ||
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<li>Wash and Ethanol Removal</li> | <li>Wash and Ethanol Removal</li> | ||
<ul> | <ul> | ||
- | <li>Add 700 | + | <li>Add 700 μL Wash Buffer (W9) with ethanol to the column</li> |
<li>Centrifuge the column at 12,000 X g for 1 minute</li> | <li>Centrifuge the column at 12,000 X g for 1 minute</li> | ||
<li>Discard the flow-through and place the column back into the wash tube</li> | <li>Discard the flow-through and place the column back into the wash tube</li> | ||
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<ul> | <ul> | ||
<li>Place the spin column in a clean 1.5-mL recovery tube</li> | <li>Place the spin column in a clean 1.5-mL recovery tube</li> | ||
- | <li>Add 75 | + | <li>Add 75 μL of preheated TE Buffer (TE) to the center of the column</li> |
<li>Incubate the column for 1 minute at room temperature</li> | <li>Incubate the column for 1 minute at room temperature</li> | ||
</ul> | </ul> | ||
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<li>The recovery tube now contains the purified plasmid DNA</li> | <li>The recovery tube now contains the purified plasmid DNA</li> | ||
<li>Discard the column</li> | <li>Discard the column</li> | ||
- | <li>Store plasmid DNA at 4 | + | <li>Store plasmid DNA at 4 °C (short-term) or store the DNA in aliquots at -20 °C (long-term)</li> |
</ul> | </ul> | ||
</ol> | </ol> | ||
- | + | <br> | |
- | Thank you to Life Technologies for this protocol. | + | <center>Thank you to <a href='http://lifetechnologies.com'>Life Technologies</a> for this protocol.</center> |
</p> | </p> |
Latest revision as of 17:50, 17 October 2014
Plasmid Miniprep Protocol
Show Table of Contents
Before starting:
- Add RNase A to Resuspension Buffer (R3) according to the instructions on the label; mix well
- Mark on the label that RNase A is added. Store Buffer R3 with RNase A at 4 °C
- Warm Lysis Buffer (L7) briefly at 37 °C to redissolve any particulate matter
- Add 100% ethanol to Wash Buffer (W9) and Wash Buffer (W10) according to the instructions on each label; mix well
- Store wash buffers with ethanol at room temperature
- Harvest
- Sediment the cells by centrifuging 1 - 5 mL of the overnight LB culture (use 1-2 x 109 E.coli cells for each sample)
- Remove all medium
- Resuspend
- Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet
- Resuspend pellet until it is homogeneous
- Lyse Note: This step is time sensitive.
- Add 250 μL Lysis Buffer (L7)
- Mix gently by inverting the capped tube five times (Do NOT vortex)
- Incubate the tube at room temperature for 5 minutes
- Precipitate
- Add 350 μL Precipitation Buffer (N4)
- Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous (Do NOT vortex)
- Centrifuge the lysate at >12,000 X g for 10 minutes
- Bind
- Load the supernatant from step 4 onto a spin column in a 2-mL wash tube
- Centrifuge the column at 12,000 X g for 1 minute
- Discard the flow-through and place the column back in the wash tube
- Wash and Ethanol Removal
- Add 700 μL Wash Buffer (W9) with ethanol to the column
- Centrifuge the column at 12,000 X g for 1 minute
- Discard the flow-through and place the column back into the wash tube
- Centrifuge the column at 12,000 X g for 1 minute
- Discard the was htube with the flow-through
- Elute
- Place the spin column in a clean 1.5-mL recovery tube
- Add 75 μL of preheated TE Buffer (TE) to the center of the column
- Incubate the column for 1 minute at room temperature
- Recover
- Centrifuge the column at 12,000 X g for 2 minutes
- The recovery tube now contains the purified plasmid DNA
- Discard the column
- Store plasmid DNA at 4 °C (short-term) or store the DNA in aliquots at -20 °C (long-term)