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<h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">DNA gel extraction</h2> | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">DNA gel extraction</h2> | ||
− | < | + | <h4> |
1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube.<br /> 2. Add a 3X sample | 1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube.<br /> 2. Add a 3X sample | ||
volume of Buffer DE-A. the weight of gel is equivalent to a 100ul volume.<br /> 3. Resuspend the gel in Buffer | volume of Buffer DE-A. the weight of gel is equivalent to a 100ul volume.<br /> 3. Resuspend the gel in Buffer | ||
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25-30ul of Eluent or deionized water to the center of the membrane. Let it stand for 1min at room temperature. | 25-30ul of Eluent or deionized water to the center of the membrane. Let it stand for 1min at room temperature. | ||
Centrifuge at 12,000xg for 1min. (pre-warming the Eluent at 65℃)<br /> | Centrifuge at 12,000xg for 1min. (pre-warming the Eluent at 65℃)<br /> | ||
− | </ | + | </h4> |
<h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">Electrophoresis</h2> | <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">Electrophoresis</h2> | ||
− | < | + | <h4>Agarose-Electrophoresis is used in order to see if the PCR product is correct and seperate DNA by the number of |
base pairs. | base pairs. | ||
<br /> ●marker was the 2 kb Plus DNA Ladder<br /> ●fill pockets with 5 µl DNA ladder or 10 µl sample volume with | <br /> ●marker was the 2 kb Plus DNA Ladder<br /> ●fill pockets with 5 µl DNA ladder or 10 µl sample volume with | ||
6x loading buffer<br /> ●running conditions: 130 V for 30-40 minutes<br /> | 6x loading buffer<br /> ●running conditions: 130 V for 30-40 minutes<br /> | ||
− | </ | + | </h4> |
<h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">Gibson assembly</h2> | <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">Gibson assembly</h2> | ||
− | < | + | <h4> |
1. Set up the reaction.<br /> V(ul)=(0.02*bp)/concentration(ng/ul) | 1. Set up the reaction.<br /> V(ul)=(0.02*bp)/concentration(ng/ul) | ||
<br /> 2. Add the fragments into Gibson system.<br /> 3. Incubate samples in a thermocycler at 50 °C for 1h. | <br /> 2. Add the fragments into Gibson system.<br /> 3. Incubate samples in a thermocycler at 50 °C for 1h. | ||
<br /> 4. Purify the product using DNA purification kit.<br /> 5. Transform the product into the competent cells | <br /> 4. Purify the product using DNA purification kit.<br /> 5. Transform the product into the competent cells | ||
of E.coli BL21 , following the transformation protocol.<br /> | of E.coli BL21 , following the transformation protocol.<br /> | ||
− | <br /> </ | + | <br /> </h4> |
<img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4> </h4> |
<img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4> </h4> |
− | < | + | <h4> </h4> |
<h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">HPLC</h2> | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">HPLC</h2> | ||
− | < | + | <h4> |
− | for acrylic acid</ | + | for acrylic acid</h4> |
<img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4><br /> Samples have to be centrifuged at 12,000xg for 5 min in order to remove solids (cells/precipitates).<br |
/> | /> | ||
− | </ | + | </h4> |
<h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">Knock out genes of E ▪ coli (MG1655)</h2> | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">Knock out genes of E ▪ coli (MG1655)</h2> | ||
− | < | + | <h4>1. Pre-chill 1.5ml and 2ml microcentrifuge tubes, deionized water, 10% glycerol. Add 1ml LB to 2ml microcentrifuge |
tubes. | tubes. | ||
<br /> 2. When OD600=0.6, incubate competent cells on ice for 20 min.<br /> 3. Transfer the competent cells to | <br /> 2. When OD600=0.6, incubate competent cells on ice for 20 min.<br /> 3. Transfer the competent cells to | ||
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each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 30℃ overnight. | each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 30℃ overnight. | ||
Position the plates with the agar side at the top, and the lid at the bottom.<br /> | Position the plates with the agar side at the top, and the lid at the bottom.<br /> | ||
− | </ | + | </h4> |
<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:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/c/c6/Sxt_%284%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4> </h4> |
<img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4><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 | ||
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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 | 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 | 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.</ | + | and fragment into Saccharomyces cerevisiae using the LiAc SS carrier DNA PEG method.</h4> |
<img src="https://static.igem.org/mediawiki/2017/e/e0/Sxt_%286%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/e/e0/Sxt_%286%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4><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. </ | + | into 100ul 20mMNaOH and mix,99° C boiling for 30min. </h4> |
<img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4> </h4> |
<img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4> </h4> |
<h2 id="section-7" style="padding-top: 100px; margin-top: -50px;">plasmid preparation</h2> | <h2 id="section-7" style="padding-top: 100px; margin-top: -50px;">plasmid preparation</h2> | ||
− | < | + | <h4>Tiangen mini plasmid kit<br /></h4> |
<h2 id="section-8" style="padding-top: 100px; margin-top: -50px;">Plasmid transformation</h2> | <h2 id="section-8" style="padding-top: 100px; margin-top: -50px;">Plasmid transformation</h2> | ||
− | < | + | <h4>1. Pipette 50µl of competent cells and 2µl of plasmid into 1.5ml tube<br /> 2. Heat shock tubes at 42°C for 30s<br |
/> 3. Incubate on ice for 2min<br /> 4. Pipette 250µl LB media to each transformation<br /> 5. Incubate at 37°C | /> 3. Incubate on ice for 2min<br /> 4. Pipette 250µl LB media to each transformation<br /> 5. Incubate at 37°C | ||
for 1h<br /> 6. Plating<br /> 7. Pick single colonies<br /> Reference: http://parts.igem.org/Help:Protocols/Transformation<br | for 1h<br /> 6. Plating<br /> 7. Pick single colonies<br /> Reference: http://parts.igem.org/Help:Protocols/Transformation<br | ||
/> | /> | ||
− | </ | + | </h4> |
<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> | ||
Line 208: | Line 208: | ||
<img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4> </h4> |
<img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4><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 | ||
30min. | 30min. | ||
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across the plate. Incubate at 37℃ overnight. Position the plates with the agar side at the top, and the lid at | across the plate. Incubate at 37℃ overnight. Position the plates with the agar side at the top, and the lid at | ||
the bottom.<br /> 6. Select single colonies for sequencing.<br /> | the bottom.<br /> 6. Select single colonies for sequencing.<br /> | ||
− | </ | + | </h4> |
<h2 id="section-10" style="padding-top: 100px; margin-top: -50px;">Reagents</h2> | <h2 id="section-10" style="padding-top: 100px; margin-top: -50px;">Reagents</h2> | ||
− | < | + | <h4>1. LB medium(lysogeny broth)<br /> The recipe for 1l LB media is as follows:<br /> Tryptone 10g/L <br /> Yeast |
extract 5g/L <br /> NaCl 10g/L<br /> 2. 0.1mM Kanamycin<br /> MW of Kanamycin:582.58<br /> Store at -20℃<br /> 3. LB plate<br /> The recipe for 1l LB plate is as follows:<br /> Tryptone 10g/L <br /> Yeast extract 5g/L <br | extract 5g/L <br /> NaCl 10g/L<br /> 2. 0.1mM Kanamycin<br /> MW of Kanamycin:582.58<br /> Store at -20℃<br /> 3. LB plate<br /> The recipe for 1l LB plate is as follows:<br /> Tryptone 10g/L <br /> Yeast extract 5g/L <br | ||
/> NaCl 10g/L<br /> 15g Agar<br /> Add appropriate amount of resistance.<br /> 4. 2YT medium<br /> The recipe | /> NaCl 10g/L<br /> 15g Agar<br /> Add appropriate amount of resistance.<br /> 4. 2YT medium<br /> The recipe | ||
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/> B:0.05mol/L KH2P04溶液<br /> 137mMNaCl,2.7mMKCl,10mMNa2HPO4,2mMKH2PO4 for 1L.<br /> | /> B:0.05mol/L KH2P04溶液<br /> 137mMNaCl,2.7mMKCl,10mMNa2HPO4,2mMKH2PO4 for 1L.<br /> | ||
− | </ | + | </h4> |
<h2 id="section-11" style="padding-top: 100px; margin-top: -50px;">References</h2> | <h2 id="section-11" style="padding-top: 100px; margin-top: -50px;">References</h2> | ||
− | < | + | <h4>http://parts.igem.org/Help:Protocols/Transformation<br /> https://2015.igem.org/Team:Aachen/Project/Overview |
<br /> http://www.zymoresearch.com/category/all-products | <br /> http://www.zymoresearch.com/category/all-products | ||
<br /> http://www.corning.com/worldwide/en/products/life-sciences/resources/brands/axygen-brand-products.html | <br /> http://www.corning.com/worldwide/en/products/life-sciences/resources/brands/axygen-brand-products.html | ||
Line 237: | Line 237: | ||
<br /> http://www.cwbiotech.com/ | <br /> http://www.cwbiotech.com/ | ||
<br /> | <br /> | ||
− | </ | + | </h4> |
<h2 id="section-12" style="padding-top: 100px; margin-top: -50px;">the LiAc SS carrier DNA PEG method</h2> | <h2 id="section-12" style="padding-top: 100px; margin-top: -50px;">the LiAc SS carrier DNA PEG method</h2> | ||
− | < | + | <h4> |
1.use a sterile pipet tip to pick Saccharomyces cerevisiae from plates,throw the tip into the tubes of appropriate medium,incubate | 1.use a sterile pipet tip to pick Saccharomyces cerevisiae from plates,throw the tip into the tubes of appropriate medium,incubate | ||
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)) | ||
Line 250: | Line 250: | ||
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. | filtrate. | ||
− | <br /> 10.System for transformation:</ | + | <br /> 10.System for transformation:</h4> |
<img src="https://static.igem.org/mediawiki/2017/7/70/Sxt_%2811%29.png" style="max-width:60%;"></a> | <img src="https://static.igem.org/mediawiki/2017/7/70/Sxt_%2811%29.png" style="max-width:60%;"></a> | ||
− | < | + | <h4><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 | ||
the agar side at the top, and the lid at the bottom.<br /> 13.Prepare plasmid for sequencing.<br /> | the agar side at the top, and the lid at the bottom.<br /> 13.Prepare plasmid for sequencing.<br /> | ||
− | </ | + | </h4> |
<h2 id="section-13" style="padding-top: 100px; margin-top: -50px;">Whole-cell catalysis</h2> | <h2 id="section-13" style="padding-top: 100px; margin-top: -50px;">Whole-cell catalysis</h2> | ||
− | < | + | <h4> |
1. Prepare sterile tubes of 5 ml of 2YT+antibiotics. Use a sterile pipet tip to pick bacteria from plates. Throw the tip | 1. Prepare sterile tubes of 5 ml of 2YT+antibiotics. Use a sterile pipet tip to pick bacteria from plates. Throw the tip | ||
into the tubes. Incubate in a rotary shaker at37℃ for 3-4h.<br /> 2. Transfer 120µl of bacteria from | into the tubes. Incubate in a rotary shaker at37℃ for 3-4h.<br /> 2. Transfer 120µl of bacteria from | ||
Line 271: | Line 271: | ||
catalysis using recombinant Escherichia coli, overexpressing adenylate cyclase[J]. Korean Journal of Chemical | catalysis using recombinant Escherichia coli, overexpressing adenylate cyclase[J]. Korean Journal of Chemical | ||
Engineering, 2013, 30(4):913-917.<br /> | Engineering, 2013, 30(4):913-917.<br /> | ||
− | </ | + | </h4> |
Revision as of 13:15, 21 October 2017