From 2009.igem.org
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| | ==Miniprep Procedure== | | ==Miniprep Procedure== |
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| - | '''Production of Cleared Lysate'''
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| - | 1. Pellet 1-10ml overnight cultures for 5 minutes
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| - | 2. Thoroughly re-suspend pellet with 250ul Cell Resuspension Solution
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| - | 3. Add 250ul Cell Lysis Solution to each sample; invert 4 times to mix
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| - | 4. Add 10ul Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature
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| - | 5. Add 350 ul Neutralizer Solution; invert 4 times to mix
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| - | 6. Centrifuge at top speed for 10 minutes at room temperature.
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| - | '''Binding of Plasmid DNA'''
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| - | 7. Insert Spin Column into Collection Tube
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| - | 8. Decant cleared lysate into Spin Column
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| - | 9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough and reinsert Column into Collection Tube.
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| - | '''Washing'''
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| - | 10. Add 750ul Wash Solution (ethanol added). Centrifugeat top speed for 1 minute. Discard flowthrough and reinsert column into Collection Tube
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| - | 11. Repeat step 10 with 250ul Wash Solution
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| - | 12. Centrifuge at top speed for 2 minutes at room temperature
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| - | '''Elution'''
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| - | 13. Transfer Spin Column to a sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column.
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| - | 14. Add 100ul of Nuclease-free Water to the Spin Column. Centrifuge at top speed for 1 minute at room temperature.
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| - | 15. Discard column and store DNA at -20'C or below.
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Revision as of 12:48, 21 October 2009
Miniprep Procedure
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