"
Team:HIT-Harbin/notebook/protocol
From 2012.igem.org
| (12 intermediate revisions not shown) | |||
| Line 17: | Line 17: | ||
<ul class="children"> | <ul class="children"> | ||
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project" title="OVERVIEW">OVERVIEW</a></li> | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project" title="OVERVIEW">OVERVIEW</a></li> | ||
| - | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part1" title=" | + | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part1" title="BIOSENSOR">BIOSENSOR</a></li> |
| - | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part2" title=" | + | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part2" title="BIOKILLER">BIOKILLER</a></li> |
| - | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part3" title=" | + | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/part3" title="BIOFILM">BIOFILM</a></li> |
| - | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/model" title=" | + | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/model" title="MODELING">MODELING</a></li> |
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/application" title="APPLICATION">APPLICATION</a></li> | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/project/application" title="APPLICATION">APPLICATION</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
| - | <li class="page_item page-item-39 "><a href=" | + | <li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/parts" title="PARTS">PARTS</a></li> |
<li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/team/safety" title="SAFETY">SAFETY </a></li> | <li class="page_item page-item-39 "><a href="https://2012.igem.org/Team:HIT-Harbin/team/safety" title="SAFETY">SAFETY </a></li> | ||
| Line 43: | Line 43: | ||
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/lecture" title="LECTURE">LECTURE</a></li> | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/lecture" title="LECTURE">LECTURE</a></li> | ||
<li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/software" title="SOFTRWARE">SOFTRWARE</a></li> | <li class="page_item page-item-136"><a href="https://2012.igem.org/Team:HIT-Harbin/humanpractice/software" title="SOFTRWARE">SOFTRWARE</a></li> | ||
| - | |||
</ul> | </ul> | ||
</li> | </li> | ||
| Line 59: | Line 58: | ||
<a>The Composition of LB liquid Medium</a> | <a>The Composition of LB liquid Medium</a> | ||
</div> | </div> | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>Yeast extact 5g</p> | <p>Yeast extact 5g</p> | ||
<p>Peptone 10g</p> | <p>Peptone 10g</p> | ||
| Line 72: | Line 71: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>Yeast extact 5g</p> | <p>Yeast extact 5g</p> | ||
<p>Peptone 10g</p> | <p>Peptone 10g</p> | ||
| Line 85: | Line 84: | ||
<div class="post-391 odd"> | <div class="post-391 odd"> | ||
<div class="post-title"> | <div class="post-title"> | ||
| - | <a> | + | <a>The Preparation of Competent Cell of Escherichia.coli</a> |
</div><!--post-title--> | </div><!--post-title--> | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
| - | <p>1. Place one colony in 3~5mL LB media, grow overnight at 37℃ | + | <p>1. Place one colony in 3~5mL LB media, grow overnight at 37℃;</p> |
<p>2. Transfer overnight DH5α culture(1:100~1:50)into 100mL LB liquid media;</p> | <p>2. Transfer overnight DH5α culture(1:100~1:50)into 100mL LB liquid media;</p> | ||
<p>3. Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (2-3 hours);</p> | <p>3. Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (2-3 hours);</p> | ||
<p>4. Place cells on ice for 10 min;</p> | <p>4. Place cells on ice for 10 min;</p> | ||
| - | <p>5. Centrifuge cells at 4℃ | + | <p>5. Centrifuge cells at 4℃ for 10 min at 3,000g;</p> |
<p>Subsequent resuspensions is done in the same bottle. Cells remain cold for the rest of the procedure: transport tubes on ice and resuspend on ice in the cold room.</p> | <p>Subsequent resuspensions is done in the same bottle. Cells remain cold for the rest of the procedure: transport tubes on ice and resuspend on ice in the cold room.</p> | ||
<p>6. Pour off media and resuspend cells in 10mL cold 0.1 M CaCl2, and incubate on ice for 30 min;</p> | <p>6. Pour off media and resuspend cells in 10mL cold 0.1 M CaCl2, and incubate on ice for 30 min;</p> | ||
| - | <p>7. Centrifuge at 4℃ | + | <p>7. Centrifuge at 4℃ for 10 min at 3,000g;</p> |
<p>8. Pour supernatant and resuspend cells (by pipetting) in 4mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 100 μL into 1.5mL centrifuge tubes placed on ice. Cells is stored at -80℃ and can be used for transformation for up to 6 months.</p> | <p>8. Pour supernatant and resuspend cells (by pipetting) in 4mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 100 μL into 1.5mL centrifuge tubes placed on ice. Cells is stored at -80℃ and can be used for transformation for up to 6 months.</p> | ||
</div><!--post-content--> | </div><!--post-content--> | ||
| Line 105: | Line 104: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>1. Add</p> | <p>1. Add</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2012/ | + | <img src="https://static.igem.org/mediawiki/2012/0/0f/Ppp1.png"></p> |
<p>2. Mix gently and spin down for a few seconds.</p> | <p>2. Mix gently and spin down for a few seconds.</p> | ||
| - | <p>3. Incubate at 37℃ | + | <p>3. Incubate at 37℃ for 1-16 hours</p> |
</div><!--post-content--> | </div><!--post-content--> | ||
</div><!--post-390--> | </div><!--post-390--> | ||
| Line 117: | Line 116: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>1. Add</p> | <p>1. Add</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2012/ | + | <img src="https://static.igem.org/mediawiki/2012/3/30/Ppp2.png"></p> |
<p>2. Mix gently and spin down for a few seconds.</p> | <p>2. Mix gently and spin down for a few seconds.</p> | ||
<p>3. Incubate at 37℃ for 1-16 hours</p> | <p>3. Incubate at 37℃ for 1-16 hours</p> | ||
| Line 129: | Line 128: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>1. Add</p> | <p>1. Add</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2012/ | + | <img src="https://static.igem.org/mediawiki/2012/f/f6/Ppp3.png"></p> |
<p>2. Mix gently and spin down for a few seconds.</p> | <p>2. Mix gently and spin down for a few seconds.</p> | ||
<p>3. Incubate at 37℃ for 1-16 hours</p> | <p>3. Incubate at 37℃ for 1-16 hours</p> | ||
| Line 141: | Line 140: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
| - | <p>1. Add | + | <p>1. Add</p> |
| - | <img src="https://static.igem.org/mediawiki/2012/ | + | <img src="https://static.igem.org/mediawiki/2012/8/84/Ppp4.png"></p> |
<p>2. Mix gently and spin down for a few seconds.</p> | <p>2. Mix gently and spin down for a few seconds.</p> | ||
<p>3. Incubate at 37℃ for 1-16 hours</p> | <p>3. Incubate at 37℃ for 1-16 hours</p> | ||
| Line 153: | Line 152: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>1. Prepare the following reaction mixture:</p> | <p>1. Prepare the following reaction mixture:</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2012/6/ | + | <img src="https://static.igem.org/mediawiki/2012/6/61/Ppp5.png"> |
<p>2. Incubate 10 min at 22℃ </p> | <p>2. Incubate 10 min at 22℃ </p> | ||
<p>3. Use up to 5μL of the mixture for transformation of 50μL of chemically competent cells.</p> | <p>3. Use up to 5μL of the mixture for transformation of 50μL of chemically competent cells.</p> | ||
| Line 165: | Line 164: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>10μL 5x buffer(Mg2+ plus)</p> | <p>10μL 5x buffer(Mg2+ plus)</p> | ||
<p>4μL dNTPs</p> | <p>4μL dNTPs</p> | ||
| Line 181: | Line 180: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>1. Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA;</p> | <p>1. Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA;</p> | ||
<p>2. Place the gel in the apparatus rig with the wells facing the negative end (black-colored);</p> | <p>2. Place the gel in the apparatus rig with the wells facing the negative end (black-colored);</p> | ||
| Line 195: | Line 194: | ||
</div><!--post-title--> | </div><!--post-title--> | ||
. | . | ||
| - | <div class="post- | + | <div class="post-excerpte"> |
<p>1. Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice;</p> | <p>1. Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice;</p> | ||
<p>2. Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel(100mg = 100μL);</p> | <p>2. Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel(100mg = 100μL);</p> | ||
Latest revision as of 05:25, 23 October 2012
Yeast extact 5g
Peptone 10g
NaCl 10g
Distilled water 1000ml pH 7.0
Range of application: Escherichia.coli
Yeast extact 5g
Peptone 10g
NaCl 10g
Agar 1-2%
Distilled water 1000ml pH 7.0
Boil the mixture in autoclave at 121℃ for 30min
Range of application: Escherichia.coli
1. Place one colony in 3~5mL LB media, grow overnight at 37℃;
2. Transfer overnight DH5α culture(1:100~1:50)into 100mL LB liquid media;
3. Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (2-3 hours);
4. Place cells on ice for 10 min;
5. Centrifuge cells at 4℃ for 10 min at 3,000g;
Subsequent resuspensions is done in the same bottle. Cells remain cold for the rest of the procedure: transport tubes on ice and resuspend on ice in the cold room.
6. Pour off media and resuspend cells in 10mL cold 0.1 M CaCl2, and incubate on ice for 30 min;
7. Centrifuge at 4℃ for 10 min at 3,000g;
8. Pour supernatant and resuspend cells (by pipetting) in 4mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 100 μL into 1.5mL centrifuge tubes placed on ice. Cells is stored at -80℃ and can be used for transformation for up to 6 months.
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Add
2. Mix gently and spin down for a few seconds.
3. Incubate at 37℃ for 1-16 hours
1. Prepare the following reaction mixture:
2. Incubate 10 min at 22℃
3. Use up to 5μL of the mixture for transformation of 50μL of chemically competent cells.
10μL 5x buffer(Mg2+ plus)
4μL dNTPs
1.0μL forward primer
1.0μL reverse primer
1.0μL template (10pg-1ng)
1.0μL DNA polymerase
ddH2O up to 50.0μL
Note: Based on primers, set an appropriate annealing tem.
Keep everything on ice and add all volumes in a PCR tube.
1. Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA;
2. Place the gel in the apparatus rig with the wells facing the negative end (black-colored);
3. Fill the rig with 1x TBE buffer;
4. Load 2μL of 1kb ladder;
5. Add 2μL of 6x loading dye to each PCR reaction tube, adn load 20μL in each well;
6. Run at 120V.
1. Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice;
2. Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel(100mg = 100μL);
3. Dissolve the gel slice using a 60μC heat block;
4. Apply the dissolved gel to the QIAquick column and centrifuge at 13,000rpm for 1 minute;
5. Discard the flow-through and repeat Step 4 until all sample has passed through the column;
6. Add 750μL of rinse buffer to the QIAquick column and centrifuge at 13,000rpm for 1 minute;
7. Add 500L of rinse buffer to the QIAquick column and centrifuge at 13,000rpm for 1 minute;
8. Wash the column with 750μL of Buffer PE and centrifuge at 13,000rpm for 1 minute;
9. Discard the flow-through and centrifuge at 13,000rpm for 2 minute to remove residual EtOH;
10. Transfer the QIAquick column to a new Eppendorf;
11. Add 50μL elution buffer to the center of the column and wait at least 2 minutes;
12. Centrifuge at 13,000rpm for 1 minute.
