Team:UEA-JIC Norwich/Methods

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<STYLE>H2 {FONT-SIZE: 21pt; COLOR: #000080}High Efficiency Transformation Protocol</h2>
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<h1>Click on the happy scientist to open the protocol
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<Br>
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<p style="color:#FFFFFF">1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes</p>
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High Efficiency Transformation Protocol
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<p style="color:#FFFFFF">2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right)</p>
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<br>
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<p style="color:#FFFFFF">3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times</p>
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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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<p style="color:#FFFFFF">4. Inoculate with DNA ( 1 μl) to the competent cells</p>
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<br>
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<p style="color:#FFFFFF">5. Place the mixture on ice for 30 minutes. Do not mix</p>
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Culture Preparation Protocol
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<p style="color:#FFFFFF">6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix</p>
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<p style="color:#FFFFFF">7. Place on ice for 5 minutes. Do not mix</p>
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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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<p style="color:#FFFFFF">8. Pipette 950 μl of room temperature SOC into the mixture</p>
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<p style="color:#FFFFFF">9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate</p>
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Glycerol Stock Solution Protocol
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<p style="color:#FFFFFF">10. Warm selection plates to 37°C</p>
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<br>
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<p style="color:#FFFFFF">11. Do serial dilutions (2-3) at 105. Mix cells, flick/invert gently</p>
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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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<p style="color:#FFFFFF">12. Spread (100 μl) on agar plates of each dilution</p>
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<p style="color:#FFFFFF">13. Incubate overnight at 37°C</p>
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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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<h2>Culture Preparation Protocol</h2>
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Gel Electrophoresis Protocol
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<p style="color:#FFFFFF">1. Select a colony by sampling it with a wooden pick</p>
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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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<p style="color:#FFFFFF">2. Place into the appropriate antibiotic LB broth</p>
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<br>
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<p style="color:#FFFFFF">3. Incubate at 37°C overnight in an Incubator Shaker</p>
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PCR Protocol
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<p style="color:#FFFFFF">4. Store at -20°C</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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<h2>Glycerol Stock Solution Protocol</h2>
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PEG Transformation Protocol
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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:#FFFFFF">1. Take 500µL of a previously prepared culture and place into a screw-top eppendorf tube</p>
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<br>
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<p style="color:#FFFFFF">2. Add 500µL of Glycerol solution (V.V 50% to make a 25% concentration</p>
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Algae Transformation using glass beads
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<p style="color:#FFFFFF">3. Place in -20°C storage</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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<h2>Qiagen miniprep Protocol</h2>
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Algae Transformation using electroporation
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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:#FFFFFF">1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. </p>
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<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>
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Site Directed Mutagenesis Protocol
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<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>
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<p style="color:#FFFFFF">4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. </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:#FFFFFF">5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting. </p>
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<p style="color:#FFFFFF">6. Centrifuge for 30–60 s. Discard the flow-through. </p>
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pGEM T-easy Vector Cloning protocol
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<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>
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<p style="color:#FFFFFF">8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. </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:#FFFFFF">9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. </p>
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<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>
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Restriction Digest protocol
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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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<h2>Gel Electrophoresis Protocol</h2>
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Poly "A" Tailing Protocol
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<p style="color:#FFFFFF">1. Measure 0.6g of agarose and add to 60 mL TAE Buffer</p>
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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:#FFFFFF">2. Mix throroughly</p>
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<p style="color:#FFFFFF">3. Heat until solution becomes clear</p>
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Ligation Protocol
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<p style="color:#FFFFFF">4. Allow to cool, then add 30µl of 1mg per ml Ethidium Bromide</p>
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<p style="color:#FFFFFF">5. Pour solution into gel tray with comb in place</p>
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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:#FFFFFF">6. Allow to set</p>
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<p style="color:#FFFFFF">7. Place in electrophoresis tank and submerge in TAE buffer</p>
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Gel Extraction Protocol
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<p style="color:#FFFFFF">8. Pipette 10 µl of ladder into left most well</p>
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<p style="color:#FFFFFF">9. Perform serial dilution of sample</p>
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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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<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>
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<p style="color:#FFFFFF">11. Run gel at 90V for 30 minutes</p>
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<p style="color:#FFFFFF">2. Visualise using UV light</p>
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<h2>PCR Protocol</h2>
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<p style="color:#FFFFFF">1. Program desired cycle into PCR machine. Cycle used was as follows:</p>
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              <p style="color:#FFFFFF">94°C        - Initial Denaturation - 120 seconds</p>
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              <p style="color:#FFFFFF">94°C        -        30 seconds</p>
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              <p style="color:#FFFFFF">58-68°C - Gradient Cycle        - 30 seconds</p>
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              <p style="color:#FFFFFF">68°C        - Final Extension        - 150 seconds</p>
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              <p style="color:#FFFFFF">68°C        -                                   300 seconds</p>
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              <p style="color:#FFFFFF">4°C          - Hold</p>
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<p style="color:#FFFFFF">Second, third and fourth stages were repeated for 30 cycles.</p>
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<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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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

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