Team:UEA-JIC Norwich/Methods

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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>
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<h1>Click on the happy scientist to open the protocol
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High Efficiency Transformation Protocol
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[[File:Happy_scientist.gif|High Efficiency Transformation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/transformation]]
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<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/glycerolstock"> Glycerol Stock Solution Protocol </a>
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Culture Preparation Protocol
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[[File:Happy_scientist.gif|Culture Preparation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/cultureprep]]
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<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/miniprep"> Qiagen Miniprep Protocol </a>
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Glycerol Stock Solution Protocol  
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[[File:Happy_scientist.gif|Glycerol Stock Solution Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/glycerolstock]]
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<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/electrophoresis"> Gel Electrophoresis Protocol </a>
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Qiagen Miniprep Protocol
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[[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>
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Gel Electrophoresis Protocol
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[[File:Happy_scientist.gif|Gel Electrophoresis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/electrophoresis]]
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<a href="https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation"> PEG Transformation protocol </a>
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PCR Protocol
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<p style="color:#000000">1. Add 9ml of 8% mannitol to a petri dish.</p>
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[[File:Happy_scientist.gif|PCR Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pcr]]
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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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<p style="color:#000000">3. Add 3ml of 2% driselase to the petri dish.</p>
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PEG Transformation Protocol
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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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[[File:Happy_scientist.gif|PEG Transformation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation]]
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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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Algae Transformation using glass beads
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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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[[File:Happy_scientist.gif|Algae Transformation using glass beads Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeglassbeads]]
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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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Algae Transformation using electroporation
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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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[[File:Happy_scientist.gif|Algae Transformation using electroporation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeelectroporation]]
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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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Site Directed Mutagenesis Protocol
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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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[[File:Happy_scientist.gif|Site Directed Mutagenesis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/sdm]]
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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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pGEM T-easy Vector Cloning protocol
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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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[[File:Happy_scientist.gif|pGEM T-easy Vector Cloning Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/vectorcloning]]
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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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Restriction Digest protocol
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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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[[File:Happy_scientist.gif|Restriction Digest Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/rdigest]]
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<h1 style="font-family:verdana;color:green">Algae Transformation Method using Glass Beads</h1>
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Poly "A" Tailing Protocol
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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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[[File:Happy_scientist.gif|Poly "A" Tailing PCR Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/polyatailing]]
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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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<p style="color:#000000">4. For all tubes resuspend cells in 750µl of TAP media.
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Ligation Protocol
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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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[[File:Happy_scientist.gif|Ligation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/ligation]]
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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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Gel Extraction Protocol
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<p style="color:#000000">9.
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[[File:Happy_scientist.gif|Gel Extraction Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/gelextract]]
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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.
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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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<p style="color:#000000">4. For all tubes resuspend cells in 250µl of TAP and sucrose media.
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<p style="color:#000000">5. Add linearised G-Luc plasmid (5µL)to the suspension and place on ice.
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<p style="color:#000000">6. Transfer 250µL of algae suspension into 1mL cuvette.
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<p style="color:#000000">7. Subject cuvettes to electroporation at 2200v/ cm and place back on ice.
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<p style="color:#000000">8. Add 500µL of starch solution to each cuvette and aspirate gently.
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<p style="color:#000000">9. Add 750µL of each sample onto an LB plate.
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<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.
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<p style="color:#000000">2. Centrifuge for five minutes at 4000rpm.
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<p style="color:#000000">3. Pour off supernatant.
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<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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Latest revision as of 21:18, 21 September 2011

University of East Anglia-JIC

UNIVERSITY OF EAST ANGLIA-JOHN INNES CENTRE

Research methodsbanner.jpg



Click on the happy scientist to open the protocol


High Efficiency Transformation Protocol
High Efficiency Transformation Protocol
Culture Preparation Protocol
Culture Preparation Protocol
Glycerol Stock Solution Protocol
Glycerol Stock Solution Protocol
Qiagen Miniprep Protocol
Qiagen Miniprep Protocol
Gel Electrophoresis Protocol
Gel Electrophoresis Protocol
PCR Protocol
PCR Protocol
PEG Transformation Protocol
PEG Transformation Protocol
Algae Transformation using glass beads
Algae Transformation using glass beads Protocol
Algae Transformation using electroporation
Algae Transformation using electroporation Protocol
Site Directed Mutagenesis Protocol
Site Directed Mutagenesis Protocol
pGEM T-easy Vector Cloning protocol
pGEM T-easy Vector Cloning Protocol
Restriction Digest protocol
Restriction Digest Protocol
Poly "A" Tailing Protocol
Poly "A" Tailing PCR Protocol
Ligation Protocol
Ligation Protocol
Gel Extraction Protocol

Gel Extraction Protocol