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| + | <p style="color:#000000"> |
| - | | + | <center> |
| - | | + | <h1>Click on the happy scientist to open the protocol |
| - | <body>
| + | </h1> |
| - | <h1 style="font-family:verdana;color:green">HIGH EFFICIENCY PROTOCOL</h1>
| + | <Br> |
| - | | + | High Efficiency Transformation Protocol |
| - | <p style="color:#FFFFFF">1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes</p> | + | <br> |
| - | <p style="color:#FFFFFF">2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right)</p> | + | [[File:Happy_scientist.gif|High Efficiency Transformation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/transformation]] |
| - | <p style="color:#FFFFFF">3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times</p> | + | <br> |
| - | <p style="color:#FFFFFF">4. Inoculate with DNA ( 1 μl) to the competent cells</p>
| + | Culture Preparation Protocol |
| - | <p style="color:#FFFFFF">5. Place the mixture on ice for 30 minutes. Do not mix</p>
| + | <br> |
| - | <p style="color:#FFFFFF">6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix</p>
| + | [[File:Happy_scientist.gif|Culture Preparation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/cultureprep]] |
| - | <p style="color:#FFFFFF">7. Place on ice for 5 minutes. Do not mix</p> | + | <br> |
| - | <p style="color:#FFFFFF">8. Pipette 950 μl of room temperature SOC into the mixture</p> | + | Glycerol Stock Solution Protocol |
| - | <p style="color:#FFFFFF">9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate</p>
| + | <br> |
| - | <p style="color:#FFFFFF">10. Warm selection plates to 37°C</p>
| + | [[File:Happy_scientist.gif|Glycerol Stock Solution Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/glycerolstock]] |
| - | <p style="color:#FFFFFF">11. Do serial dilutions (2-3) at 105. Mix cells, flick/invert gently</p>
| + | <br> |
| - | <p style="color:#FFFFFF">12. Spread (100 μl) on agar plates of each dilution</p>
| + | Qiagen Miniprep Protocol |
| - | <p style="color:#FFFFFF">13. Incubate overnight at 37°C</p> | + | <br> |
| - | | + | [[File:Happy_scientist.gif|Qiagen Miniprep Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/miniprep]] |
| - | | + | <br> |
| - | | + | Gel Electrophoresis Protocol |
| - | <body> | + | <br> |
| - | <h1 style="font-family:verdana;color:green">CULTURE PREPARATION PROTOCOL</h1>
| + | [[File:Happy_scientist.gif|Gel Electrophoresis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/electrophoresis]] |
| - | | + | <br> |
| - | <p style="color:#FFFFFF">1. Select a colony by sampling it with a wooden pick</p>
| + | PCR Protocol |
| - | <p style="color:#FFFFFF">2. Place into the appropriate antibiotic LB broth</p>
| + | <br> |
| - | <p style="color:#FFFFFF">3. Incubate at 37°C overnight in an Incubator Shaker</p>
| + | [[File:Happy_scientist.gif|PCR Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pcr]] |
| - | <p style="color:#FFFFFF">4. Store at -20°C</p>
| + | <br> |
| - | | + | PEG Transformation Protocol |
| - | | + | <br> |
| - | | + | [[File:Happy_scientist.gif|PEG Transformation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/pegtransformation]] |
| - | | + | <br> |
| - | <body> | + | Algae Transformation using glass beads |
| - | <h1 style="font-family:verdana;color:green">GLYCEROL STOCK SOLUTION PROTOCOL</h1> | + | <br> |
| - | | + | [[File:Happy_scientist.gif|Algae Transformation using glass beads Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeglassbeads]] |
| - | <p style="color:#FFFFFF">1. Take 500µL of a previously prepared culture and place into a screw-top eppendorf tube</p>
| + | <br> |
| - | <p style="color:#FFFFFF">2. Add 500µL of Glycerol solution (V.V 50% to make a 25% concentration</p>
| + | Algae Transformation using electroporation |
| - | <p style="color:#FFFFFF">3. Place in -20°C storage</p>
| + | <br> |
| - | | + | [[File:Happy_scientist.gif|Algae Transformation using electroporation Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/algaeelectroporation]] |
| - | | + | <br> |
| - | | + | Site Directed Mutagenesis Protocol |
| - | <body> | + | <br> |
| - | <h1 style="font-family:verdana;color:green">QIAGEN MINIPREP PROTOCOL</h1> | + | [[File:Happy_scientist.gif|Site Directed Mutagenesis Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/sdm]] |
| - | | + | <br> |
| - | <p style="color:#FFFFFF">1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. </p>
| + | pGEM T-easy Vector Cloning protocol |
| - | <p style="color:#FFFFFF">2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns blue. </p>
| + | <br> |
| - | <p style="color:#FFFFFF">3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns colorless. </p>
| + | [[File:Happy_scientist.gif|pGEM T-easy Vector Cloning Protocol |link=https://2011.igem.org/Team:UEA-JIC_Norwich/vectorcloning]] |
| - | <p style="color:#FFFFFF">4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. </p>
| + | <br> |
| - | <p style="color:#FFFFFF">5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting. </p> | + | Restriction Digest protocol |
| - | <p style="color:#FFFFFF">6. Centrifuge for 30–60 s. Discard the flow-through. </p>
| + | <br> |
| - | <p style="color:#FFFFFF">7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is only required when using endA+ or other bacteria strains with high nuclease activity or carbohydrate content (see QIAprep Miniprep Handbookfor more details) </p>
| + | [[File:Happy_scientist.gif|Restriction Digest Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/rdigest]] |
| - | <p style="color:#FFFFFF">8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. </p>
| + | <br> |
| - | <p style="color:#FFFFFF">9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. </p>
| + | Poly "A" Tailing Protocol |
| - | <p style="color:#FFFFFF">10. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min. </p> | + | <br> |
| - | | + | [[File:Happy_scientist.gif|Poly "A" Tailing PCR Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/polyatailing]] |
| - | | + | <br> |
| - | <body> | + | Ligation Protocol |
| - | <h1 style="font-family:verdana;color:green">GEL ELECTROPHORESIS PROTOCOL</h1>
| + | <br> |
| - | | + | [[File:Happy_scientist.gif|Ligation Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/ligation]] |
| - | <p style="color:#FFFFFF">1. Measure 0.6g of agarose and add to 60 mL TAE Buffer</p>
| + | <br> |
| - | <p style="color:#FFFFFF">2. Mix throroughly</p>
| + | Gel Extraction Protocol |
| - | <p style="color:#FFFFFF">3. Heat until solution becomes clear</p> | + | <br> |
| - | <p style="color:#FFFFFF">4. Allow to cool, then add 30µl of 1mg per ml Ethidium Bromide</p>
| + | [[File:Happy_scientist.gif|Gel Extraction Protocol|link=https://2011.igem.org/Team:UEA-JIC_Norwich/gelextract]] |
| - | <p style="color:#FFFFFF">5. Pour solution into gel tray with comb in place</p>
| + | |
| - | <p style="color:#FFFFFF">6. Allow to set</p>
| + | |
| - | <p style="color:#FFFFFF">7. Place in electrophoresis tank and submerge in TAE buffer</p> | + | |
| - | <p style="color:#FFFFFF">8. Pipette 10 µl of ladder into left most well</p>
| + | |
| - | <p style="color:#FFFFFF">9. Perform serial dilution of sample</p>
| + | |
| - | <p style="color:#FFFFFF">10. Add 9 µl of sample to 1 µl of loading dye and place in wells, noting position of each sample</p>
| + | |
| - | <p style="color:#FFFFFF">11. Run gel at 90V for 30 minutes</p>
| + | |
| - | <p style="color:#FFFFFF">2. Visualise using UV light</p> | + | |
| - | | + | |
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| - | <body> | + | |
| - | <h1 style="font-family:verdana;color:green">PCR PROTOCOL</h1>
| + | |
| - | | + | |
| - | <p style="color:#FFFFFF">1. Program desired cycle into PCR machine. Cycle used was as follows:</p>
| + | |
| - | | + | |
| - | <p style="color:#FFFFFF">94°C - Initial Denaturation - 120 seconds</p>
| + | |
| - | <p style="color:#FFFFFF">94°C - 30 seconds</p>
| + | |
| - | <p style="color:#FFFFFF">58-68°C - Gradient Cycle - 30 seconds</p>
| + | |
| - | <p style="color:#FFFFFF">68°C - Final Extension - 150 seconds</p>
| + | |
| - | <p style="color:#FFFFFF">68°C - 300 seconds</p>
| + | |
| - | <p style="color:#FFFFFF">4°C - Hold</p>
| + | |
| - |
| + | |
| - | <p style="color:#FFFFFF">Second, third and fourth stages were repeated for 30 cycles.</p> | + | |
| - | <p style="color:#FFFFFF">2. Three serial dilutions were conducted, yielding concentrations of 1:10, 1:100, and 1:1000. This was carried out in order to find the optimum concentration of template DNA to use, as we didn't know the concentration yielded from the Miniprep Protocol used to extract the plasmid. We conducted a gradient cycle because even though we knew the annealing temperatures of the primers we'd designed, we wanted to make sure we had the optimum temperature range.</p>
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| - | <h1 style="font-family:verdana;color:green">PEG Transformation</h1>
| + | |
| - | | + | |
| - | <p style="color:#FFFFFF">1. Add 9ml of 8% mannitol to a petri dish.</p>
| + | |
| - | <p style="color:#FFFFFF">2. Using a spatula, put 7 day old moss from 2-3 PPNH4 plates in the petri dish containing the mannitol.</p>
| + | |
| - | <p style="color:#FFFFFF">3. Add 3ml of 2% driselase to the petri dish.</p> | + | |
| - | <p style="color:#FFFFFF">4. Incubate the petri dish at room temperature with gentle shaking for 1 hour.</p>
| + | |
| - | <p style="color:#FFFFFF">5. Filter the protoplast suspension through a 100µm mesh.</p>
| + | |
| - | <p style="color:#FFFFFF">6. Spin the filtered suspension at 250g for 5 minutes.Remove the supernatant.
| + | |
| - | <p style="color:#FFFFFF">7. Resuspend the protoplasts very gently with 500µl of 8%mannitol.</p>
| + | |
| - | <p style="color:#FFFFFF">8. Add 9.5ml 8% mannitol in the culture tube. Make sure the protoplasts are fully suspended.</p>
| + | |
| - | <p style="color:#FFFFFF">9. Repeat the filtration and re-suspension steps (6,7,8) two more times.</p>
| + | |
| - | <p style="color:#FFFFFF">10. Take 10µl of the protoplast solution and count the protoplasts using a haemocytometer.</p> | + | |
| - | <p style="color:#FFFFFF">11. Multiply the number of protoplasts in as 16 square area by 10,000 to obtain the amount of protoplastspts ml.</p> | + | |
| - | <p style="color:#FFFFFF">12. Spin the protoplast solution at 250g for 5 minutes. Remove the supernatant.
| + | |
| - | <p style="color:#FFFFFF">13. Re-suspend protoplasts in MMg solution at the concentration 1.6 million protoplasts/ml./p>
| + | |
| - | <p style="color:#FFFFFF">14. Incubate the protoplast suspension at room temperature for 20 minutes.</p>
| + | |
| - | <p style="color:#FFFFFF">15. Add 600µl of protoplast suspension into a culture tube containing 60µg DNA. Swirl the tube gently.</p>
| + | |
| - | <p style="color:#FFFFFF">16. Add 700µl of PEG/Ca solution into the protoplast/DNA mixture. Swirl the tube gently until all the mixture homogeneous.</p> | + | |
| - | <p style="color:#FFFFFF">17. Incubate the mixture at room temperature for 30 minutes.</p>
| + | |
| - | <p style="color:#FFFFFF">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> | + | |
| - | <p style="color:#FFFFFF">19. With a spatula remove any air bubbles trapped between the cellophane and the plate.</p>
| + | |
| - | <p style="color:#FFFFFF">20. Dilute the mixture with 3ml of W5 solution.</p>
| + | |
| - | <p style="color:#FFFFFF">21. Spin the mixture at 250g for 5 minutes. Remove the supernatant.</p>
| + | |
| - | <p style="color:#FFFFFF">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>
| + | |
| - | <p style="color:#FFFFFF"> Growth chamber is set to 16hrs light 8hrs dark cycle.</p>
| + | |
| - | <p style="color:#FFFFFF"> Move the cellophane onto a fresh selection plate 4 days after transformation.</p>
| + | |
| - | </body>
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| - | </html>
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