Team:UQ-Australia/Notebook/Miniprep procedure

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==Transformation Procedure 2==
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==Minoprep Procedure==
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1. Thaw competent cells on ice. Place number of required polypropylene tubes on ice.
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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. Gently mix cells, then aliquot 100ul competent cells into chilled polypropylene tubes. Add 1.7 ul of B-mercaptoethanol. Gently stir every 2 minutes for 10 minutes.
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2. Thoroughly re-suspend pellet with 250ul Cell Resuspension Solution
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3. Add 1-10ng of plasmid DNA, moving the pipette through cells while dispersing. Gently tap tubes to mix.
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3. Add 250ul Cell Lysis Solution to each sample; invert 4 times to mix
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4. Incubate cells on ice for 30 minutes
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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. Heat-shock cells for 30 seconds in 42'C waterbath. DO NOT SHAKE
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5. Add 350 ul Neutralizer Solution; invert 4 times to mix
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6. Place on ice for 2 minutes
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6. Centrifuge at top speed for 10 minutes at room temperature.
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7. Add 900ul of room temperature SOC medium + glucose
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'''Binding of Plasmid DNA'''
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7. Insert Spin Column into Collection Tube
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8. Shake at 225rpm (37'C) for 1 hour
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8. Decant cleared lysate into Spin Column
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9. During the 1 hour incubation take agar plates out of fridge and incubate in 37'C incubator for 30 minutes
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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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10. Spread 3 agar plates: 50ul, 100ul, 500ul
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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. Incubate at 37'C overnight
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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 06:32, 2 September 2009

Minoprep Procedure

Production of Cleared Lysate 1. Pellet 1-10ml overnight cultures for 5 minutes

2. Thoroughly re-suspend pellet with 250ul Cell Resuspension Solution

3. Add 250ul Cell Lysis Solution to each sample; invert 4 times to mix

4. Add 10ul Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature

5. Add 350 ul Neutralizer Solution; invert 4 times to mix

6. Centrifuge at top speed for 10 minutes at room temperature.

Binding of Plasmid DNA 7. Insert Spin Column into Collection Tube

8. Decant cleared lysate into Spin Column

9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough and reinsert Column into Collection Tube.

Washing 10. Add 750ul Wash Solution (ethanol added). Centrifugeat top speed for 1 minute. Discard flowthrough and reinsert column into Collection Tube

11. Repeat step 10 with 250ul Wash Solution

12. Centrifuge at top speed for 2 minutes at room temperature

Elution 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.

14. Add 100ul of Nuclease-free Water to the Spin Column. Centrifuge at top speed for 1 minute at room temperature.

15. Discard column and store DNA at -20'C or below.

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