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| - | <a href="https://2011.igem.org/Team:UEA-JIC_Norwich/transformation"> High Efficiency Transformation Protocol </a>
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| - | <a href="https://2011.igem.org/Team:UEA-JIC_Norwich/cultureprep"> Culture Preparation Protocol </a> | + | <p style="color:#000000"> |
| | + | <center> |
| | + | <h1>Click on the happy scientist to open the protocol |
| | + | </h1> |
| | + | <Br> |
| | + | High Efficiency Transformation Protocol |
| | <br> | | <br> |
| | + | [[File:Happy_scientist.gif|High Efficiency Transformation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/transformation]] |
| | <br> | | <br> |
| - | <a href="https://2011.igem.org/Team:UEA-JIC_Norwich/glycerolstock"> Glycerol Stock Solution Protocol </a>
| + | Culture Preparation Protocol |
| | <br> | | <br> |
| | + | [[File:Happy_scientist.gif|Culture Preparation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/cultureprep]] |
| | <br> | | <br> |
| - | <a href="https://2011.igem.org/Team:UEA-JIC_Norwich/miniprep"> Qiagen Miniprep Protocol </a>
| + | Glycerol Stock Solution Protocol |
| | <br> | | <br> |
| | + | [[File:Happy_scientist.gif|Glycerol Stock Solution Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/glycerolstock]] |
| | <br> | | <br> |
| - | <a href="https://2011.igem.org/Team:UEA-JIC_Norwich/electrophoresis"> Gel Electrophoresis Protocol </a>
| + | Qiagen Miniprep Protocol |
| | <br> | | <br> |
| | + | [[File:Happy_scientist.gif|Qiagen Miniprep Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/miniprep]] |
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| - | <a href="https://2011.igem.org/Team:UEA-JIC_Norwich/pcr"> PCR protocol </a>
| + | Gel Electrophoresis Protocol |
| | <br> | | <br> |
| | + | [[File:Happy_scientist.gif|Gel Electrophoresis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/electrophoresis]] |
| | <br> | | <br> |
| - | <a href="https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation"> PEG Transformation protocol </a> | + | PCR Protocol |
| - | | + | <br> |
| - | <p style="color:#000000">1. Add 9ml of 8% mannitol to a petri dish.</p>
| + | [[File:Happy_scientist.gif|PCR Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pcr]] |
| - | <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>
| + | <br> |
| - | <p style="color:#000000">3. Add 3ml of 2% driselase to the petri dish.</p>
| + | PEG Transformation Protocol |
| - | <p style="color:#000000">4. Incubate the petri dish at room temperature with gentle shaking for 1 hour.</p> | + | <br> |
| - | <p style="color:#000000">5. Filter the protoplast suspension through a 100µm mesh.</p> | + | [[File:Happy_scientist.gif|PEG Transformation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation]] |
| - | <p style="color:#000000">6. Spin the filtered suspension at 250g for 5 minutes.Remove the supernatant.
| + | <br> |
| - | <p style="color:#000000">7. Resuspend the protoplasts very gently with 500µl of 8%mannitol.</p>
| + | Algae Transformation using glass beads |
| - | <p style="color:#000000">8. Add 9.5ml 8% mannitol in the culture tube. Make sure the protoplasts are fully suspended.</p>
| + | <br> |
| - | <p style="color:#000000">9. Repeat the filtration and re-suspension steps (6,7,8) two more times.</p>
| + | [[File:Happy_scientist.gif|Algae Transformation using glass beads Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeglassbeads]] |
| - | <p style="color:#000000">10. Take 10µl of the protoplast solution and count the protoplasts using a haemocytometer.</p> | + | <br> |
| - | <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> | + | Algae Transformation using electroporation |
| - | <p style="color:#000000">12. Spin the protoplast solution at 250g for 5 minutes. Remove the supernatant.
| + | <br> |
| - | <p style="color:#000000">13. Re-suspend protoplasts in MMg solution at the concentration 1.6 million protoplasts/ml.</p>
| + | [[File:Happy_scientist.gif|Algae Transformation using electroporation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeelectroporation]] |
| - | <p style="color:#000000">14. Incubate the protoplast suspension at room temperature for 20 minutes.</p>
| + | <br> |
| - | <p style="color:#000000">15. Add 600µl of protoplast suspension into a culture tube containing 60µg DNA. Swirl the tube gently.</p>
| + | Site Directed Mutagenesis Protocol |
| - | <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> | + | <br> |
| - | <p style="color:#000000">17. Incubate the mixture at room temperature for 30 minutes.</p>
| + | [[File:Happy_scientist.gif|Site Directed Mutagenesis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/sdm]] |
| - | <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> | + | <br> |
| - | <p style="color:#000000">19. With a spatula remove any air bubbles trapped between the cellophane and the plate.</p>
| + | pGEM T-easy Vector Cloning protocol |
| - | <p style="color:#000000">20. Dilute the mixture with 3ml of W5 solution.</p>
| + | <br> |
| - | <p style="color:#000000">21. Spin the mixture at 250g for 5 minutes. Remove the supernatant.</p>
| + | [[File:Happy_scientist.gif|pGEM T-easy Vector Cloning Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/vectorcloning]] |
| - | <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> | + | Restriction Digest protocol |
| - | <p style="color:#000000"> Move the cellophane onto a fresh selection plate 4 days after transformation.</p>
| + | <br> |
| - | </body>
| + | [[File:Happy_scientist.gif|Restriction Digest Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/rdigest]] |
| - | | + | <br> |
| - | <h1 style="font-family:verdana;color:green">Algae Transformation Method using Glass Beads</h1>
| + | Poly "A" Tailing Protocol |
| - | <p style="color:#000000">1. Transfer 50ml of algae culture into four 50ml screw top tubes. | + | <br> |
| - | <p style="color:#000000">2. Place all four tubes in the eppendorf centrifuge at 800rpm for 9 minutes at 20-22°C.
| + | [[File:Happy_scientist.gif|Poly "A" Tailing PCR Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/polyatailing]] |
| - | <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.
| + | <br> |
| - | <p style="color:#000000">4. For all tubes resuspend cells in 750µl of TAP media.
| + | Ligation Protocol |
| - | <p style="color:#000000">5. Wash pre prepared beads in ethanol removing excess gently.
| + | <br> |
| - | <p style="color:#000000">6. Wash beads approximately 2-3 times in autoclaved distilled water.
| + | [[File:Happy_scientist.gif|Ligation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/ligation]] |
| - | <p style="color:#000000">7. Place approximately half the beads in a 1.5ml eppendorf tube for the control. | + | <br> |
| - | <p style="color:#000000">8. Resuspend both tubes with 200µL of distilled water.
| + | Gel Extraction Protocol |
| - | <p style="color:#000000">9. | + | <br> |
| - | <p style="color:#000000">10.
| + | [[File:Happy_scientist.gif|Gel Extraction Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/gelextract]] |
| - | <p style="color:#000000">11.
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| - | <p style="color:#000000">12.
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| - | <p style="color:#000000">13.
| + | |
| - | <p style="color:#000000">14. | + | |
| - | <p style="color:#000000">15.
| + | |
| - | <p style="color:#000000">16. | + | |
| - | <p style="color:#000000">17.
| + | |
| - | <p style="color:#000000">18.
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| - | <p style="color:#000000">19.
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| - | <p style="color:#000000">20.
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| - | <p style="color:#000000">21.
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| - | <p style="color:#000000">22.
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| - | <h1 style="font-family:verdana;color:green">Algae Transformation Method using Electroporation</h1>
| + | |
| - | <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">3. Immediately pour contents of tubes into glass beaker and keep tubes upside down so no unwanted suspension mixes with the pellet. | + | |
| - | <p style="color:#000000">4. For all tubes resuspend cells in 250µl of TAP and sucrose media.
| + | |
| - | <p style="color:#000000">5. Add linearised G-Luc plasmid (5µL)to the suspension and place on ice.
| + | |
| - | <p style="color:#000000">6. Transfer 250µL of algae suspension into 1mL cuvette.
| + | |
| - | <p style="color:#000000">7. Subject cuvettes to electroporation at 2200v/ cm and place back on ice.
| + | |
| - | <p style="color:#000000">8. Add 500µL of starch solution to each cuvette and aspirate gently.
| + | |
| - | <p style="color:#000000">9. Add 750µL of each sample onto an LB plate.
| + | |
| - | <p style="color:#000000">10.Parafilm the lid and plate to seal and place in a light incubator.
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| - | <h1 style="font-family:verdana;color:green">Starch Preparation</h1>
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| - | <p style="color:#000000">1. Wash starch powder with 10mL distilled water and resuspend. | + | |
| - | <p style="color:#000000">2. Centrifuge for five minutes at 4000rpm.
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| - | <p style="color:#000000">3. Pour off supernatant. | + | |
| - | <p style="color:#000000">4. Resuspend with 500µL Ethanol.
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| - | <p style="color:#000000">5. Centrifuge for five minutes at 4000rpm.
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| - | <p style="color:#000000">6. Resuspend in 500µL TAP.
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| - | <p style="color:#000000">7. Centrifuge for five minutes at 4000rpm, pour off supernatant, and repeat once.
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| - | <p style="color:#000000">8. Resuspend in 500µL TAP with sucrose.
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| - | </html>
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