Difference between revisions of "Team:RHIT/Protocol"
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− | < | + | <h2 class="attn">Protocols</h2> |
− | + | ||
− | + | ||
<ul> | <ul> | ||
− | <li class="Title" id="QIAPREP">QIAprep Spin Miniprep Kit</li> | + | <li class="Title" id="QIAPREP" onmouseover="changecursor('QIAPREP')">QIAprep Spin Miniprep Kit</li> |
− | <ol id="QIAPREPSTEP"> | + | <ol id="QIAPREPSTEP" style="display:none"> |
<li class="steps"> Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature.</li> | <li class="steps"> Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature.</li> | ||
<li class="steps">Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.</li> | <li class="steps">Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.</li> | ||
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<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>Reference: Qiagen; Netherlands; Product #27104</b></p> | ||
</ol> | </ol> | ||
<br> | <br> | ||
− | <li class="Title" id="NEBuilder">NEBuilder HiFi DNA Assembly Reaction Protocol</li> | + | <li class="Title" id="NEBuilder" onmouseover="changecursor('NEBuilder')">NEBuilder HiFi DNA Assembly Reaction Protocol</li> |
− | <ol id="NEBuilderSTEP"> | + | <ol id="NEBuilderSTEP" style="display:none"> |
<li class="steps">Set up the following reaction on ice (for 2-3 fragment assembly):</li> | <li class="steps">Set up the following reaction on ice (for 2-3 fragment assembly):</li> | ||
<ul> | <ul> | ||
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</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> | ||
</ol> | </ol> | ||
<br> | <br> | ||
− | <li class="Title" id="BOILING">Boiling Prep Protocol</li> | + | <li class="Title" id="BOILING" onmouseover="changecursor('BOILING')">Boiling Prep Protocol</li> |
− | <ol id="BOILINGSTEP"> | + | <ol id="BOILINGSTEP" style="display:none"> |
<li class="steps">Spin down cells.</li> | <li class="steps">Spin down cells.</li> | ||
<li class="steps">Resuspend cells in 350 µl STET buffer.</li> | <li class="steps">Resuspend cells in 350 µl STET buffer.</li> | ||
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<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> | ||
</ol> | </ol> | ||
<br> | <br> | ||
− | <li class="Title" id="NEB">NEB High Efficiency Transformation Protocol</li> | + | <li class="Title" id="NEB" onmouseover="changecursor('NEB')">NEB High Efficiency Transformation Protocol</li> |
− | <ol id="NEBSTEP"> | + | <ol id="NEBSTEP" style="display:none"> |
<li class="steps">Thaw a tube of NE 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.</li> | <li class="steps">Thaw a tube of NE 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.</li> | ||
<li class="steps">Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. <b>Do not vortex</b>.</li> | <li class="steps">Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. <b>Do not vortex</b>.</li> | ||
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<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> | ||
</ol> | </ol> | ||
<br> | <br> | ||
− | <li class="Title" id="QIAquick">QIAquick Gel Extraction Kit</li> | + | <li class="Title" id="QIAquick" onmouseover="changecursor('QIAquick')">QIAquick Gel Extraction Kit</li> |
− | <ol id="QIAquickSTEP"> | + | <ol id="QIAquickSTEP" style="display:none"> |
<li class="steps">Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li> | <li class="steps">Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li> | ||
<li class="steps">Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 µl).</li> | <li class="steps">Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 µl).</li> | ||
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<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> | ||
</ol> | </ol> | ||
<br> | <br> | ||
− | <li class="Title" id="Ethanol">Ethanol Precipitation</li> | + | <li class="Title" id="Ethanol" onmouseover="changecursor('Ethanol')">Ethanol Precipitation</li> |
− | <ol id="EthanolSTEP"> | + | <ol id="EthanolSTEP" style="display:none"> |
<li class="steps">Estimate the volume of the DNA solution.</li> | <li class="steps">Estimate the volume of the DNA solution.</li> | ||
<li class="steps">Adjust the concentration of monovalent cations either by dilution with TE (pH 8.0) if the DNA solution contains a high concentration of salts of by addition of one of the salt solutions shown in Table A8-1. (If the volume of the final solution is 400 µl or less, carry out precipitation in a single microcentrifuge tube. Larger volumes can be divided among several microfuge tubes, or the DNA can be precipitated and centrifuged in tubes that will fit in a medium-speed centrifuge or ultracentrifuge.)</li> | <li class="steps">Adjust the concentration of monovalent cations either by dilution with TE (pH 8.0) if the DNA solution contains a high concentration of salts of by addition of one of the salt solutions shown in Table A8-1. (If the volume of the final solution is 400 µl or less, carry out precipitation in a single microcentrifuge tube. Larger volumes can be divided among several microfuge tubes, or the DNA can be precipitated and centrifuged in tubes that will fit in a medium-speed centrifuge or ultracentrifuge.)</li> | ||
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<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> | ||
</ol> | </ol> | ||
<br> | <br> | ||
− | <li class="Title" id="Yeast">Yeast Transformation (From Breeden Lab)</li> | + | <li class="Title" id="Yeast" onmouseover="changecursor('Yeast')">Yeast Transformation (From Breeden Lab)</li> |
− | <ol id="YeastSTEP"> | + | <ol id="YeastSTEP" style="display:none"> |
<li class="steps">Pick colonies and inoculate cultures in YPD and grow overnight.</li> | <li class="steps">Pick colonies and inoculate cultures in YPD and grow overnight.</li> | ||
<li class="steps">Measure OD<sub>600</sub> nm and dilute to OD<sub>600</sub> nm = 0.3 in 50 ml.</li> | <li class="steps">Measure OD<sub>600</sub> nm and dilute to OD<sub>600</sub> nm = 0.3 in 50 ml.</li> | ||
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<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> | ||
+ | <br><br><br> | ||
+ | <img style="padding-left:5%;width:90%" src="https://static.igem.org/mediawiki/2015/7/74/RHIT_SIDEBANNER.png"> | ||
<br><br><br> | <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)