Difference between revisions of "Team:RHIT/Protocol"
(13 intermediate revisions by 3 users not shown) | |||
Line 53: | Line 53: | ||
</script> | </script> | ||
</head> | </head> | ||
− | |||
− | |||
<h2 class="attn">Protocols</h2> | <h2 class="attn">Protocols</h2> | ||
Line 74: | Line 72: | ||
<li class="steps">Centrifuge for 1 min to remove residual wash buffer.</li> | <li class="steps">Centrifuge for 1 min to remove residual wash buffer.</li> | ||
<li class="steps">Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB or water to the center of the QIAprep spin column, let it stand for 1 min, and centrifuge for 1 min.</li> | <li class="steps">Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB or water to the center of the QIAprep spin column, let it stand for 1 min, and centrifuge for 1 min.</li> | ||
− | <p><b>Qiagen; Netherlands; Product #27104</b></p> | + | <p><b>Reference: Qiagen; Netherlands; Product #27104</b></p> |
</ol> | </ol> | ||
<br> | <br> | ||
Line 92: | Line 90: | ||
</li> | </li> | ||
<li class="steps">Transform NEB 5-alpha Competent E. coli cells with 2 µl of the assembled product.</li> | <li class="steps">Transform NEB 5-alpha Competent E. coli cells with 2 µl of the assembled product.</li> | ||
− | <p><b>Reference:</b>New England Biolabs; Ipswich, MA; Product #E5520S</p> | + | <p><b>Reference: </b>New England Biolabs; Ipswich, MA; Product #E5520S</p> |
</ol> | </ol> | ||
<br> | <br> | ||
Line 109: | Line 107: | ||
<li class="steps">Wash pellet with 70% ethanol.</li> | <li class="steps">Wash pellet with 70% ethanol.</li> | ||
<li class="steps">Resuspend in water or TE.</li> | <li class="steps">Resuspend in water or TE.</li> | ||
− | <p><b>Reference:</b>Sambrook, J., & Russell, D. (2001). <i>Molecular Cloning: A Laboratory Manual</i> (3rd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.</p> | + | <p><b>Reference: </b>Sambrook, J., & Russell, D. (2001). <i>Molecular Cloning: A Laboratory Manual</i> (3rd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.</p> |
</ol> | </ol> | ||
<br> | <br> | ||
Line 125: | Line 123: | ||
<li class="steps">Warm selection plates to 37ºC.</li> | <li class="steps">Warm selection plates to 37ºC.</li> | ||
<li class="steps">Spread 50-100 µl of transformation onto a selection plate and incubate overnight at 37ºC. Alternatively, incubate at 30ºC for 24-36 hours or 25ºC for 48 hours.</li> | <li class="steps">Spread 50-100 µl of transformation onto a selection plate and incubate overnight at 37ºC. Alternatively, incubate at 30ºC for 24-36 hours or 25ºC for 48 hours.</li> | ||
− | <p> <b>Reference:</b>New England Biolabs; Ipswich, MA</p> | + | <p> <b>Reference: </b>New England Biolabs; Ipswich, MA</p> |
</ol> | </ol> | ||
<br> | <br> | ||
Line 145: | Line 143: | ||
<li class="steps">To elute DNA, add 50 µl Buffer EB or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.</li> | <li class="steps">To elute DNA, add 50 µl Buffer EB or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.</li> | ||
<li class="steps">If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</li> | <li class="steps">If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</li> | ||
− | <p><b>Reference:</b>Qiagen; Netherlands; Product #28704</p> | + | <p><b>Reference: </b>Qiagen; Netherlands; Product #28704</p> |
</ol> | </ol> | ||
<br> | <br> | ||
Line 161: | Line 159: | ||
<li class="steps">Store the open tube on the bench at room temperature until the last traces of fluid have evaporated.</li> | <li class="steps">Store the open tube on the bench at room temperature until the last traces of fluid have evaporated.</li> | ||
<li class="steps">Dissolve DNA pellet in the desired volume of buffer (usually TE). Rinse the walls of the tube with the buffer.</li> | <li class="steps">Dissolve DNA pellet in the desired volume of buffer (usually TE). Rinse the walls of the tube with the buffer.</li> | ||
− | <p> <b>Reference:</b>Sambrook, J., & Russell, D. (2001). <i>Molecular Cloning: A Laboratory Manual</i> (3rd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.</p> | + | <p> <b>Reference: </b>Sambrook, J., & Russell, D. (2001). <i>Molecular Cloning: A Laboratory Manual</i> (3rd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.</p> |
</ol> | </ol> | ||
<br> | <br> | ||
Line 183: | Line 181: | ||
<li class="steps">Spin and resuspend cells in 0.5 ml 1 x TE.</li> | <li class="steps">Spin and resuspend cells in 0.5 ml 1 x TE.</li> | ||
<li class="steps">Place 100 - 200 µl per plate and incubate at 30℃ for three days.</li> | <li class="steps">Place 100 - 200 µl per plate and incubate at 30℃ for three days.</li> | ||
+ | <p>http://research.fhcrc.org/breeden/en/methods.html</p> | ||
</ol> | </ol> | ||
</ul> | </ul> | ||
Line 188: | Line 187: | ||
<br><br><br> | <br><br><br> | ||
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img style="padding-left:5%;width:90%" src="https://static.igem.org/mediawiki/2015/7/74/RHIT_SIDEBANNER.png"> |
+ | <br><br><br> | ||
</div></div> | </div></div> | ||
</html> | </html> |
Latest revision as of 22:43, 18 September 2015
Protocols
- QIAprep Spin Miniprep Kit
- NEBuilder HiFi DNA Assembly Reaction Protocol
- Boiling Prep Protocol
- NEB High Efficiency Transformation Protocol
- QIAquick Gel Extraction Kit
- Ethanol Precipitation
- Yeast Transformation (From Breeden Lab)