Team:Tokyo Metropolitan/Project/Fiber/Protocol
From 2010.igem.org
(Difference between revisions)
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- | <font size="3"><span style="text-decoration:underline">Material</font></span><br> | + | <font size="3"><span style="text-decoration:underline">Material</font></span> |
- | <font size="3"><span style="text-decoration:underline">Equipment</font></span><br> | + | <ul> |
- | <font size="3"><span style="text-decoration:underline">Procedure</font></span><br> | + | <li>1% agarose gel |
+ | <ul> | ||
+ | <li>agarose S 3g | ||
+ | <li>TAE buffer 300ml | ||
+ | <li>ethidium bromide 15µl | ||
+ | </ul> | ||
+ | <li>1×TAE buffer | ||
+ | <li>10×Loading buffer 5µl | ||
+ | <li>template DNA(PCR production) | ||
+ | </ul> | ||
+ | <br> | ||
+ | <font size="3"><span style="text-decoration:underline">Equipment</font></span> | ||
+ | <ul> | ||
+ | <li>microwave | ||
+ | <li>gel box | ||
+ | <li>flask(500ml) | ||
+ | </ul> | ||
+ | <br> | ||
+ | <font size="3"><span style="text-decoration:underline">Procedure</font></span> | ||
+ | <ol> | ||
+ | <li>Measure out 3g agarose into a beaker with the 300ml of TAE buffer.Microwave until the agarose is fully melted(5minutes×3). | ||
+ | <li>Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel). At this point add your DNA stain, e.g., ethidium bromide 15µl. The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles. | ||
+ | <li>While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side. Make sure it is sealed well or the gel will leak. | ||
+ | <li>Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid. | ||
+ | <li>If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten. | ||
+ | <li>set agarose gel and add TAE buffer in gel box. | ||
+ | <li>mix DNA and Loading buffer and then put in well them(marker sets another well). | ||
+ | <li>load DNA at 100V for two third of entire(about 15minutes). | ||
+ | <li>image the consequence of electrophoreses. | ||
+ | </ol> | ||
+ | <br> | ||
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- | <font size="3"><span style="text-decoration:underline">Material</font></span>< | + | <font size="3"><span style="text-decoration:underline">Material</font></span> |
- | + | <ul> | |
- | + | <li>QIAGEN(gel extraction kit) | |
- | + | <ul> | |
- | + | <li>QG buffer 300µl | |
- | + | <li>PE buffer700µl | |
- | + | <li>EB buffer 50µl | |
- | <font size="3"><span style="text-decoration:underline">Equipment</font></span>< | + | <li>tube for column |
- | + | </ul> | |
- | + | </ul> | |
- | + | <br> | |
- | + | <font size="3"><span style="text-decoration:underline">Equipment</font></span> | |
- | <font size="3"><span style="text-decoration:underline">Procedure</font></span>< | + | <ul> |
- | + | <li>centrifuge | |
- | + | <li>heating plate | |
- | + | <li>pipet | |
- | + | <li>pipet tip | |
- | + | </ul> | |
- | + | <br> | |
- | + | <font size="3"><span style="text-decoration:underline">Procedure</font></span> | |
- | + | <ol> | |
- | + | <li>cut gel of electrophoreses | |
- | + | <li>add pieces of gel to tubes | |
- | + | <li>take 300µl QG buffer into tubes and dissolve at 50°C | |
- | + | <li>add a solution of QG buffer and gel to tubes for column | |
- | + | <li>centrifuge 15000rpm/1min | |
+ | <li>throw “flow-thru” away and take 700µl PE buffer | ||
+ | <li>centrifuge 15000rpm/1min | ||
+ | <li>throw flow-thru away | ||
+ | <li>centrifuge 15000rpm/1min | ||
+ | <li>change tube for column | ||
+ | <li>add 50µl of EB buffer (aim to center of tube) | ||
+ | <li>centrifuge 15000rpm/1min | ||
+ | </ol> | ||
</td></tr></table></center></html> | </td></tr></table></center></html> |
Revision as of 09:22, 29 August 2010
E.coli Fiber Project Protocol
Contents |
Grow up a culture of A.xylinus in media
Grow up a culture of A.xylinus in media recommend by Open Wet Ware
Material
Equipment
Procedure
Note
|
Grow up a culture of A.xylinus in media recommend by JCM
Material
Equipment
Procedure
Note
|
Grow up a culture of E.coli
Material
Equipment
Procedure
|
Direct PCR
Material
Equipment
Procedure
|
Electrophoreses PCR productions
Material
Equipment
Procedure
|
DNA purification from agarose gel with QIAGEN
Material
Equipment
Procedure
|