Team:UEA-JIC Norwich/Methods

From 2011.igem.org

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<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation"> PEG Transformation protocol </a>
<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation"> PEG Transformation protocol </a>
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<p style="color:#000000">1. Add 9ml of 8% mannitol to a petri dish.</p>
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<p style="color:#000000">2. Using a spatula, put 7 day old moss from 2-3 PPNH4 plates in the petri dish containing the mannitol.</p>
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<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">3. Add 3ml of 2% driselase to the petri dish.</p>
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<p style="color:#000000">4. Incubate the petri dish at room temperature with gentle shaking for 1 hour.</p>
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<p style="color:#000000">5. Filter the protoplast suspension through a 100µm mesh.</p>
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<p style="color:#000000">6. Spin the filtered suspension at 250g for 5 minutes.Remove the supernatant.
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<p style="color:#000000">7. Resuspend the protoplasts very gently with 500µl of 8%mannitol.</p>
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<p style="color:#000000">8. Add 9.5ml 8% mannitol in the culture tube. Make sure the protoplasts are fully suspended.</p>
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<p style="color:#000000">9. Repeat the filtration and re-suspension steps (6,7,8) two more times.</p>
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<p style="color:#000000">10. Take 10µl of the protoplast solution and count the protoplasts using a haemocytometer.</p>
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<p style="color:#000000">11. Multiply the number of protoplasts in as 16 square area by 10,000 to obtain the amount of protoplastspts ml.</p>
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<p style="color:#000000">12. Spin the protoplast solution at 250g for 5 minutes. Remove the supernatant.
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<p style="color:#000000">13. Re-suspend protoplasts in MMg solution at the concentration 1.6 million protoplasts/ml.</p>
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<p style="color:#000000">14. Incubate the protoplast suspension at room temperature for 20 minutes.</p>
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<p style="color:#000000">15. Add 600µl of protoplast suspension into a culture tube containing 60µg DNA. Swirl the tube gently.</p>
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<p style="color:#000000">16. Add 700µl of PEG/Ca solution into the protoplast/DNA mixture. Swirl the tube gently until all the mixture homogeneous.</p>
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<p style="color:#000000">17. Incubate the mixture at room temperature for 30 minutes.</p>
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<p style="color:#000000">18. During this waiting period cover PRM-B plates with 80mm cellophane discs. Allow the cellophane to hydrate on the plate surface for at least 5 minutes.</p>
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<p style="color:#000000">19. With a spatula remove any air bubbles trapped between the cellophane and the plate.</p>
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<p style="color:#000000">20. Dilute the mixture with 3ml of W5 solution.</p>
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<p style="color:#000000">21. Spin the mixture at 250g for 5 minutes. Remove the supernatant.</p>
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<p style="color:#000000">22. Re-suspend protoplasts in melted 2ml of PRM-T. Plate 1ml of re-suspended protoplasts per PRM-B plate covered by cellophane. Wrap the plates with micropore tape and keep in a 25°C growth chamber.</p>
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<p style="color:#000000"> Growth chamber is set to 16hrs light 8hrs dark cycle.</p>
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<p style="color:#000000"> Move the cellophane onto a fresh selection plate 4 days after transformation.</p>
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<h1 style="font-family:verdana;color:green">Algae Transformation Method using Glass Beads</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 14:48, 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

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 750µl of TAP media.

5. Wash pre prepared beads in ethanol removing excess gently.

6. Wash beads approximately 2-3 times in autoclaved distilled water.

7. Place approximately half the beads in a 1.5ml eppendorf tube for the control.

8. Resuspend both tubes with 200µL of distilled water.

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Algae Transformation Method using Electroporation

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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