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Team:CSU Fort Collins/Notebook/Protocols=Miniprep
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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> | ||
| Line 756: | Line 756: | ||
<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> | ||
Revision as of 17:47, 9 October 2014
Plasmid Miniprep Protocol
Show Table of Contents
PLASMID MINIPREP
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 DEGC
- Warm Lysis Buffer (L7) briefly at 37 DEGC 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 DEGC (short-term) or store the DNA in aliquots at -20 DEGC (long-term)
