Team:UQ-Australia/Notebook/Project1

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(Miniprep Procedure)
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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.
 

Revision as of 12:48, 21 October 2009

Miniprep Procedure