Team:Hong Kong-CUHK/4.3
From 2012.igem.org
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<p><strong>Part 1: BioBrick Construction</strong></p> | <p><strong>Part 1: BioBrick Construction</strong></p> | ||
- | <p><strong>Step 1: Polymerase Chain Reaction (PCR)</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 1: Polymerase Chain Reaction (PCR)</strong></p> |
<p>A DNA amplification process to make the target sequence into linear DNA copies</p> | <p>A DNA amplification process to make the target sequence into linear DNA copies</p> | ||
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DNA templates should be added at last, and thermocycle should start immediately after adding all the materials</p> | DNA templates should be added at last, and thermocycle should start immediately after adding all the materials</p> | ||
- | <p><strong>Step 2: DAN Digestion by Restriction Enzyme (Restriction Cut)</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 2: DAN Digestion by Restriction Enzyme (Restriction Cut)</strong></p> |
<p>Restriction cut can be used and repeated for different purposes: create a sticky end of the DNA, make blunt end of DNA, or make linear plasmid into circular form.</p> | <p>Restriction cut can be used and repeated for different purposes: create a sticky end of the DNA, make blunt end of DNA, or make linear plasmid into circular form.</p> | ||
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Glycerol can be added to prevent further undesirable DNA digestion</p> | Glycerol can be added to prevent further undesirable DNA digestion</p> | ||
- | <p><strong>Step 3: DNA Purification</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 3: DNA Purification</strong></p> |
<p>Removing excess nucleotides, enzymes and ions in the solution with desired 100 bp - 10 kb DNA </p> | <p>Removing excess nucleotides, enzymes and ions in the solution with desired 100 bp - 10 kb DNA </p> | ||
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The Chinese University of Hong Kong School of Life Science.Version 2.2. </p> | The Chinese University of Hong Kong School of Life Science.Version 2.2. </p> | ||
- | <p><strong>Step 4: DNA Purification from Gel</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 4: DNA Purification from Gel</strong></p> |
<p>After checking the band size through gel electrophoresis, enzymes, ions, nucleotides, needs to be removed from solution for the target DNA (70 bp - 10 kb)</p> | <p>After checking the band size through gel electrophoresis, enzymes, ions, nucleotides, needs to be removed from solution for the target DNA (70 bp - 10 kb)</p> | ||
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Both DNA purifications give high possibility of correct ligation later. But gel purification allows the selection of target DNA sequence from the other DNA fragments while PCR purification only lowers the contamination. </p> | Both DNA purifications give high possibility of correct ligation later. But gel purification allows the selection of target DNA sequence from the other DNA fragments while PCR purification only lowers the contamination. </p> | ||
- | <p><strong>Step 5: T4 DNA Ligation</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 5: T4 DNA Ligation</strong></p> |
<p>Linking the sticky ends or blunt end of DNA segments after digestion to make linear or circular plasmid</p> | <p>Linking the sticky ends or blunt end of DNA segments after digestion to make linear or circular plasmid</p> | ||
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Reaction takes place over night at - 4 or 16 。C or for 2 hours at 22 to 25。 C</p> | Reaction takes place over night at - 4 or 16 。C or for 2 hours at 22 to 25。 C</p> | ||
- | <p><strong>Step 6: Bacterial Transformation</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 6: Bacterial Transformation</strong></p> |
<p>Opening pores on the membrane of cell by heat shock or chemical to allow circular DNA plasmid to enter</p> | <p>Opening pores on the membrane of cell by heat shock or chemical to allow circular DNA plasmid to enter</p> | ||
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The Chinese University of Hong Kong School of Life Science.Version 2.2.</p> | The Chinese University of Hong Kong School of Life Science.Version 2.2.</p> | ||
- | <p><strong>Step 7: Biobrick Amplification in Plasmid Form</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 7: Biobrick Amplification in Plasmid Form</strong></p> |
<p>Amplifying the desire DNA plasmid by growing the colonies from the spread plates with antibiotic to select the expect bacteria which contains the backbone making the bacteria to be resistant to a particular antibiotic.</p> | <p>Amplifying the desire DNA plasmid by growing the colonies from the spread plates with antibiotic to select the expect bacteria which contains the backbone making the bacteria to be resistant to a particular antibiotic.</p> | ||
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Storage of the bacteria cell with desired plasmid: 800 μl of cell + 200 μl of 80% sterile glycerol, and they should be stored in -80。C freezer. </p> | Storage of the bacteria cell with desired plasmid: 800 μl of cell + 200 μl of 80% sterile glycerol, and they should be stored in -80。C freezer. </p> | ||
- | <p><strong>Step 8: Plasmid DNA Extraction</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 8: Plasmid DNA Extraction</strong></p> |
<p>Material: miniprep plasmid DNA extraction kit</p> | <p>Material: miniprep plasmid DNA extraction kit</p> | ||
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<p><strong>Part 2: Characterization of BioBrick</strong></p> | <p><strong>Part 2: Characterization of BioBrick</strong></p> | ||
- | <p><strong>Step 1: DNA Size Determination</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 1: DNA Size Determination</strong></p> |
<p>Through comparing DNA ladder as the reference with your DNA or plasmid, you can know whether you have the desired DNA or plasmid. This step is normally done to ensure the quality of DNA after PCR, restriction enzyme cut, ligation, and extraction. </p> | <p>Through comparing DNA ladder as the reference with your DNA or plasmid, you can know whether you have the desired DNA or plasmid. This step is normally done to ensure the quality of DNA after PCR, restriction enzyme cut, ligation, and extraction. </p> | ||
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100ng of DNA is needed to be loaded and appeared in the image of the gel</p> | 100ng of DNA is needed to be loaded and appeared in the image of the gel</p> | ||
- | <p><strong>Step 2: Preparation of DNA for Nucleotide Sequence Determination</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 2: Preparation of DNA for Nucleotide Sequence Determination</strong></p> |
<p>Material: Backward and forward primers sequence the DNA and result is analysis by sequencing machine and shown in a computer with the software. This is the genotypic method to identify the bacteria, and in this case, to see whether it matches your BioBrick design. </p> | <p>Material: Backward and forward primers sequence the DNA and result is analysis by sequencing machine and shown in a computer with the software. This is the genotypic method to identify the bacteria, and in this case, to see whether it matches your BioBrick design. </p> | ||
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This can be sent and done by some biotechnology companies around your area. </p> | This can be sent and done by some biotechnology companies around your area. </p> | ||
- | <p><strong>Step 3: Determination of Fluorescent Signal</strong></p> | + | <p class="GREEN" style="font-size: 14px"><strong>Step 3: Determination of Fluorescent Signal</strong></p> |
<p>Fluorescent plate reader is used to measure and calculate the fluorescent signal expressed from fluorescence-fusion protein | <p>Fluorescent plate reader is used to measure and calculate the fluorescent signal expressed from fluorescence-fusion protein | ||
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The Chinese University of Hong Kong School of Life Science.Version 2.2.</p> | The Chinese University of Hong Kong School of Life Science.Version 2.2.</p> | ||
- | + | <p class="GREEN" style="font-size: 10px"><a href="https://2012.igem.org/Team:Hong_Kong-CUHK/HP_GUIDE"><u>BACK TO CONTENT</u></a></p> | |
Latest revision as of 17:24, 26 October 2012
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CH4.3 Stage 3 – Make your BioBrick Here are the descriptions of one of the traditional way to make a BioBrick. General materials are listed to be prepared before the lab starts, and some points should be noted in the process. This session can be used as your check list before you are familiar with the actual protocol from different kits, but please take note that variation always exists and understanding the protocol before start is helpful as well as necessary for the desired result. Do not be upset with negative result. Protocols vary among the different designs of the BioBrick, and asides from the one described above, there are other ways to make a BioBrick.
Part 1: BioBrick Construction Step 1: Polymerase Chain Reaction (PCR) A DNA amplification process to make the target sequence into linear DNA copies Material: dNTPs, water, DNA templates, forward and reverse primers, heat stable DNA polymerase and its corresponding buffer Note: Step 2: DAN Digestion by Restriction Enzyme (Restriction Cut) Restriction cut can be used and repeated for different purposes: create a sticky end of the DNA, make blunt end of DNA, or make linear plasmid into circular form. Material: water , DNA template, restriction enzyme, 100X BSA buffer, 10X NEWB buffer, and 37。C incubator or dry bath Note: Step 3: DNA Purification Removing excess nucleotides, enzymes and ions in the solution with desired 100 bp - 10 kb DNA Material: use PCR Purification Kit Note: Source: Loo J. 2012. iGEM Hong_Kong-CUHK 2012 Wet-lab Training Manual. Step 4: DNA Purification from Gel After checking the band size through gel electrophoresis, enzymes, ions, nucleotides, needs to be removed from solution for the target DNA (70 bp - 10 kb) Material: use gel clean kit, distilled water, and 60。C dry bath Note: Step 5: T4 DNA Ligation Linking the sticky ends or blunt end of DNA segments after digestion to make linear or circular plasmid Materials: T4 DNA ligase, 10X T4 DNA buffer, ddH2O, restriction enzyme cut, 16。C dry bath or - 4。C fridge Note: Step 6: Bacterial Transformation Opening pores on the membrane of cell by heat shock or chemical to allow circular DNA plasmid to enter Material: aseptic technique, cell, LB plates, plasmid DNA, LB media or SOC media, incubator Notes: Source: Loo J. 2012. iGEM Hong_Kong-CUHK 2012 Wet-lab Training Manual. Step 7: Biobrick Amplification in Plasmid Form Amplifying the desire DNA plasmid by growing the colonies from the spread plates with antibiotic to select the expect bacteria which contains the backbone making the bacteria to be resistant to a particular antibiotic. Material: autoclaved tooth pick, LB media, antibiotic, snap cap, and 37。C shaker Notes: Step 8: Plasmid DNA Extraction Material: miniprep plasmid DNA extraction kit Notes: Source: Loo J. 2012. iGEM Hong_Kong-CUHK 2012 Wet-lab Training Manual. Part 2: Characterization of BioBrick Step 1: DNA Size Determination Through comparing DNA ladder as the reference with your DNA or plasmid, you can know whether you have the desired DNA or plasmid. This step is normally done to ensure the quality of DNA after PCR, restriction enzyme cut, ligation, and extraction. Material: Agarose gel, TAE buffer, gel imager, computer, DNA ladder, 6X or 10X DNA loading dye, DNA staining dye. Notes: Step 2: Preparation of DNA for Nucleotide Sequence Determination Material: Backward and forward primers sequence the DNA and result is analysis by sequencing machine and shown in a computer with the software. This is the genotypic method to identify the bacteria, and in this case, to see whether it matches your BioBrick design. Notes: Step 3: Determination of Fluorescent Signal Fluorescent plate reader is used to measure and calculate the fluorescent signal expressed from fluorescence-fusion protein Material: 96-well plate, standard fluorescence, and micro-plate reader Notes: Source: Loo J. 2012. iGEM Hong_Kong-CUHK 2012 Wet-lab Training Manual. |
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