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− | <div class="batu" style="background: url('https://static.igem.org/mediawiki/2017/f/fe/Npu-background.png') no-repeat fixed;"> | + | <div class="batu" style="background: url('https://static.igem.org/mediawiki/2017/f/fe/Npu-background.png') no-repeat fixed; overflow: hidden;"> |
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<div class="container"> | <div class="container"> | ||
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<img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:50%;"></a> | ||
<p> </p> | <p> </p> | ||
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<img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:50%;"></a> | ||
<p> </p> | <p> </p> | ||
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</p> | </p> | ||
<h2 id="section-6" style="padding-top: 100px; margin-top: -50px;">Knock out the genes of Saccharomyces cerevisiae with Crispr-Cas9</h2> | <h2 id="section-6" style="padding-top: 100px; margin-top: -50px;">Knock out the genes of Saccharomyces cerevisiae with Crispr-Cas9</h2> | ||
− | + | ||
<img src="https://static.igem.org/mediawiki/2017/c/c6/Sxt_%284%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/c/c6/Sxt_%284%29.png" style="max-width:50%;"></a> | ||
<p> </p> | <p> </p> | ||
− | + | ||
<img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width:50%;"></a> | ||
<p><br /> 2. Purify the PCR product with a DNA purification kit.<br /> 3. Add the appropriate amount of DMT enzyme, | <p><br /> 2. Purify the PCR product with a DNA purification kit.<br /> 3. Add the appropriate amount of DMT enzyme, | ||
hold for one hour at 37 ° C.<br /> 4. transform the DNA into competent cells.<br /> 50ul competent cell + 15ul | hold for one hour at 37 ° C.<br /> 4. transform the DNA into competent cells.<br /> 50ul competent cell + 15ul | ||
purified DNA,incubate on ice for 30min,heat shock 45s,incubate on ice for 2min,add LB medium and incubate for | purified DNA,incubate on ice for 30min,heat shock 45s,incubate on ice for 2min,add LB medium and incubate for | ||
− | 1h.<br /> 5. Pipet 100ul from each tube onto the plate with resistance, and spread the mixture evenly across | + | 1h. |
− | + | <br /> 5. Pipet 100ul from each tube onto the plate with resistance, and spread the mixture evenly across the | |
− | + | plate. Incubate for 12h. Position the plates with the agar side at the top, and the lid at the bottom.<br /> 6. use a sterile pipet tip to pick Saccharomyces cerevisiae from plates,throw the tip into the tubes of 5 ml | |
− | + | of LB + antibiotics,incubate in a rotary shaker. Prepare plasmid with kit for sequencing.<br /> 7. Transfer plasmid | |
− | + | and fragment into Saccharomyces cerevisiae using the LiAc SS carrier DNA PEG method.</p> | |
<img src="https://static.igem.org/mediawiki/2017/e/e0/Sxt_%286%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/e/e0/Sxt_%286%29.png" style="max-width:50%;"></a> | ||
<p><br /> 8. Prepare the template: use a sterile toothpick to pick Saccharomyces cerevisiae from plates,put the toothpick | <p><br /> 8. Prepare the template: use a sterile toothpick to pick Saccharomyces cerevisiae from plates,put the toothpick | ||
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<img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width:50%;"></a> | ||
<p> </p> | <p> </p> | ||
− | + | ||
<img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width:50%;"></a> | ||
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<h2 id="section-9" style="padding-top: 100px; margin-top: -50px;">Point mutation</h2> | <h2 id="section-9" style="padding-top: 100px; margin-top: -50px;">Point mutation</h2> | ||
− | + | ||
− | + | ||
<img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width:50%;"></a> | ||
<p> </p> | <p> </p> | ||
− | + | ||
<img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width:50%;"></a> | <img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width:50%;"></a> | ||
<p><br /> 2. Purify the PCR product with a DNA purification kit.<br /> 3. Add the appropriate amount of DMT enzyme, | <p><br /> 2. Purify the PCR product with a DNA purification kit.<br /> 3. Add the appropriate amount of DMT enzyme, | ||
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in a rotary shaker for 12h.<br /> 2.measure OD600, transfer x(x=(50×0.2)/(OD600×dilution ratio)) | in a rotary shaker for 12h.<br /> 2.measure OD600, transfer x(x=(50×0.2)/(OD600×dilution ratio)) | ||
ml Saccharomyces cerevisiae into 50ml YPAD.<br /> 3.incubate for 4-5h to make OD600 reaches 0.8-0.9.<br /> 4.boil | ml Saccharomyces cerevisiae into 50ml YPAD.<br /> 3.incubate for 4-5h to make OD600 reaches 0.8-0.9.<br /> 4.boil | ||
− | ssDNA.<br /> 5.Centrifuge at 3000g for 5 min. Discard the filtrate. Repeat washes with 25ml deionized water twice.<br | + | ssDNA. |
+ | <br /> 5.Centrifuge at 3000g for 5 min. Discard the filtrate. Repeat washes with 25ml deionized water twice.<br | ||
/> 6.Transfer the cells to 1.5 mL centrifuge tube. Add 1ml of deionized water, resuspend the cells gently.<br | /> 6.Transfer the cells to 1.5 mL centrifuge tube. Add 1ml of deionized water, resuspend the cells gently.<br | ||
/> 7.Centrifuge at 13000rpm for 30s. Discard the filtrate.<br /> 8.Add 1ml of deionized water, resuspend the | /> 7.Centrifuge at 13000rpm for 30s. Discard the filtrate.<br /> 8.Add 1ml of deionized water, resuspend the | ||
cells. Pipet 100ul into each 1.5 mL centrifuge tube.<br /> 9.Centrifuge using a Mini Centrifuge. Discard the | cells. Pipet 100ul into each 1.5 mL centrifuge tube.<br /> 9.Centrifuge using a Mini Centrifuge. Discard the | ||
− | filtrate.<br /> 10.System for transformation:</p> | + | filtrate. |
− | + | <br /> 10.System for transformation:</p> | |
+ | <img src="https://static.igem.org/mediawiki/2017/7/70/Sxt_%2811%29.png" style="max-width:50%;"></a> | ||
<p><br /> 11.Incubate at 30℃ for 20min.<br /> 12.42℃ heat shock for 40min. pipet 100ul from each tube onto the appropriate | <p><br /> 11.Incubate at 30℃ for 20min.<br /> 12.42℃ heat shock for 40min. pipet 100ul from each tube onto the appropriate | ||
plate, and spread the mixture evenly across the plate. Incubate at 30℃ for 2-3 days. Position the plates with | plate, and spread the mixture evenly across the plate. Incubate at 30℃ for 2-3 days. Position the plates with | ||
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Revision as of 13:12, 21 October 2017