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of E.coli BL21 , following the transformation protocol.<br /> | of E.coli BL21 , following the transformation protocol.<br /> | ||
<br /> </p> | <br /> </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:75%;"></a> |
<p> </p> | <p> </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:75%;"></a> |
<p> </p> | <p> </p> | ||
<p> </p> | <p> </p> | ||
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<p> | <p> | ||
for acrylic acid</p> | for acrylic acid</p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:75%;"></a> |
<p><br /> Samples have to be centrifuged at 12,000xg for 5 min in order to remove solids (cells/precipitates).<br | <p><br /> Samples have to be centrifuged at 12,000xg for 5 min in order to remove solids (cells/precipitates).<br | ||
/> | /> | ||
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<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: | + | <img src="https://static.igem.org/mediawiki/2017/c/c6/Sxt_%284%29.png" style="max-width:75%;"></a> |
<p> </p> | <p> </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width:75%;"></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 | ||
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of LB + antibiotics,incubate in a rotary shaker. Prepare plasmid with kit for sequencing.<br /> 7. Transfer plasmid | 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> | 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: | + | <img src="https://static.igem.org/mediawiki/2017/e/e0/Sxt_%286%29.png" style="max-width:75%;"></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 | ||
into 100ul 20mMNaOH and mix,99° C boiling for 30min. </p> | into 100ul 20mMNaOH and mix,99° C boiling for 30min. </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width:75%;"></a> |
<p> </p> | <p> </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width:75%;"></a> |
<p> </p> | <p> </p> | ||
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− | <img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width:75%;"></a> |
<p> </p> | <p> </p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width:75%;"></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 5μl digested DNA into competent cells DH5α, incubate on ice for | hold for one hour at 37 ° C.<br /> 4. Transform 5μl digested DNA into competent cells DH5α, incubate on ice for | ||
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filtrate. | filtrate. | ||
<br /> 10.System for transformation:</p> | <br /> 10.System for transformation:</p> | ||
− | <img src="https://static.igem.org/mediawiki/2017/7/70/Sxt_%2811%29.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2017/7/70/Sxt_%2811%29.png" style="max-width:75%;"></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 |
Revision as of 13:12, 21 October 2017