Team:UQ-Australia/Notebook/Miniprep procedure

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==Transformation Procedure 2==
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==Miniprep 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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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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1. Pellet 1-10ml overnight cultures for 5 minutes
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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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2. Thoroughly re-suspend pellet with 250ul Cell Resuspension Solution
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4. Incubate cells on ice for 30 minutes
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3. Add 250ul Cell Lysis Solution to each sample; invert 4 times to mix
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5. Heat-shock cells for 30 seconds in 42'C waterbath. DO NOT SHAKE
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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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6. Place on ice for 2 minutes
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5. Add 350 ul Neutralizer Solution; invert 4 times to mix
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7. Add 900ul of room temperature SOC medium + glucose
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6. Centrifuge at top speed for 10 minutes at room temperature.
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8. Shake at 225rpm (37'C) for 1 hour
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'''Binding of Plasmid DNA'''
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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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7. Insert Spin Column into Collection Tube
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10. Spread 3 agar plates: 50ul, 100ul, 500ul
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8. Decant cleared lysate into Spin Column
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11. Incubate at 37'C overnight
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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.

Latest revision as of 06:33, 2 September 2009

Miniprep 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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