Team:UQ-Australia/Notebook/Experiment1

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==Miniprep Procedure==
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==Gel Extraction for PCR product of AG43==
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'''Production of Cleared Lysate'''
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Running PCR products of Ag43 collected from reaction1 (named PCR1) and 2 (named PCR2) (Click HERE for further details on 06/10/09 ) on 1% agarose gel.
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Click HERE for the gel image. (File 4)
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1. Pellet 1-10ml overnight cultures for 5 minutes
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Follow the procedure described in Quick Gel Extraction Kit Manual (Click HERE for more details)
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Result: Nanodrop  of gel extraction ‘s final products:
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2. Thoroughly re-suspend pellet with 250ul Cell Resuspension Solution
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Sample name -- Concentration (ng/uL) -- 260/280
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PCR1 -- 40.04 -- 1.99
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3. Add 250ul Cell Lysis Solution to each sample; invert 4 times to mix
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PCR2 -- 27.05 -- 1.96
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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 12:32, 20 October 2009

Gel Extraction for PCR product of AG43

Running PCR products of Ag43 collected from reaction1 (named PCR1) and 2 (named PCR2) (Click HERE for further details on 06/10/09 ) on 1% agarose gel. Click HERE for the gel image. (File 4)

Follow the procedure described in Quick Gel Extraction Kit Manual (Click HERE for more details) Result: Nanodrop of gel extraction ‘s final products: Sample name -- Concentration (ng/uL) -- 260/280 PCR1 -- 40.04 -- 1.99 PCR2 -- 27.05 -- 1.96

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