Team:UEA-JIC Norwich/Methods

From 2011.igem.org

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<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/algaeglassbeads"> Algae Transformation using glass beads protocol </a>
<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/algaeglassbeads"> Algae Transformation using glass beads protocol </a>
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<p style="color:#000000">1. Transfer 50ml of algae culture into four 50ml screw top tubes.
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<p style="color:#000000">2. Place all four tubes in the eppendorf centrifuge at 800rpm for 9 minutes at 20-22°C.
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<p style="color:#000000">3. Immediately pour contents of tubes into glass beaker and keep tubes upside down so no unwanted suspension mixes with the pellet.
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<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/algaeelectroporation"> Algae Transformation using electroporation protocol </a>
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<p style="color:#000000">4. For all tubes resuspend cells in 750µl of TAP media.
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<p style="color:#000000">5. Wash pre prepared beads in ethanol removing excess gently.
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<p style="color:#000000">6. Wash beads approximately 2-3 times in autoclaved distilled water.
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<p style="color:#000000">7. Place approximately half the beads in a 1.5ml eppendorf tube for the control.
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<p style="color:#000000">8. Resuspend both tubes with 200µL of distilled water.
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<h1 style="font-family:verdana;color:green">Algae Transformation Method using Electroporation</h1>
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<p style="color:#000000">1. Transfer 50ml of algae culture into four 50ml screw top tubes.
<p style="color:#000000">1. Transfer 50ml of algae culture into four 50ml screw top tubes.
<p style="color:#000000">2. Place all four tubes in the eppendorf centrifuge at 800rpm for 9 minutes at 20-22°C.
<p style="color:#000000">2. Place all four tubes in the eppendorf centrifuge at 800rpm for 9 minutes at 20-22°C.

Revision as of 14:51, 21 September 2011

University of East Anglia-JIC

UNIVERSITY OF EAST ANGLIA-JOHN INNES CENTRE

High Efficiency Transformation Protocol

Culture Preparation Protocol

Glycerol Stock Solution Protocol

Qiagen Miniprep Protocol

Gel Electrophoresis Protocol

PCR protocol

PEG Transformation protocol

Algae Transformation using glass beads protocol

Algae Transformation using electroporation protocol

1. Transfer 50ml of algae culture into four 50ml screw top tubes.

2. Place all four tubes in the eppendorf centrifuge at 800rpm for 9 minutes at 20-22°C.

3. Immediately pour contents of tubes into glass beaker and keep tubes upside down so no unwanted suspension mixes with the pellet.

4. For all tubes resuspend cells in 250µl of TAP and sucrose media.

5. Add linearised G-Luc plasmid (5µL)to the suspension and place on ice.

6. Transfer 250µL of algae suspension into 1mL cuvette.

7. Subject cuvettes to electroporation at 2200v/ cm and place back on ice.

8. Add 500µL of starch solution to each cuvette and aspirate gently.

9. Add 750µL of each sample onto an LB plate.

10.Parafilm the lid and plate to seal and place in a light incubator.

Starch Preparation

1. Wash starch powder with 10mL distilled water and resuspend.

2. Centrifuge for five minutes at 4000rpm.

3. Pour off supernatant.

4. Resuspend with 500µL Ethanol.

5. Centrifuge for five minutes at 4000rpm.

6. Resuspend in 500µL TAP.

7. Centrifuge for five minutes at 4000rpm, pour off supernatant, and repeat once.

8. Resuspend in 500µL TAP with sucrose.

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