Team:Caltech/Project/Methods and Methods
From 2014.igem.org
(Difference between revisions)
m |
|||
(11 intermediate revisions not shown) | |||
Line 43: | Line 43: | ||
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; modeling.style.color='white'" onMouseOut="this.bgColor='white'; modeling.style.color='black'" bgColor=white> | <td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; modeling.style.color='white'" onMouseOut="this.bgColor='white'; modeling.style.color='black'" bgColor=white> | ||
- | <a id='modeling' href="https://2014.igem.org/Team:Caltech/ | + | <a id='modeling' href="https://2014.igem.org/Team:Caltech/TXTL"style="color:#000000"> TXTL Promoter Characterization</a></td> |
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; notebook.style.color='white'" onMouseOut="this.bgColor='white'; notebook.style.color='black'" bgColor=white> | <td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; notebook.style.color='white'" onMouseOut="this.bgColor='white'; notebook.style.color='black'" bgColor=white> | ||
Line 71: | Line 71: | ||
<tr> | <tr> | ||
<td width="20%" valign="top"> | <td width="20%" valign="top"> | ||
- | <a href = "https://2014.igem.org/Team:Caltech/Project"> | + | <a href = "https://2014.igem.org/Team:Caltech/Project">Project Overview</a> |
<br><br> | <br><br> | ||
<a href = "https://2014.igem.org/Team:Caltech/Project/Details">Project Details</a> | <a href = "https://2014.igem.org/Team:Caltech/Project/Details">Project Details</a> | ||
Line 79: | Line 79: | ||
<a href = "https://2014.igem.org/Team:Caltech/Project/Experiments">The Experiments</a> | <a href = "https://2014.igem.org/Team:Caltech/Project/Experiments">The Experiments</a> | ||
<br><br> | <br><br> | ||
- | <a href = "https://2014.igem.org/Team:Caltech/Project/Results">Results | + | <a href = "https://2014.igem.org/Team:Caltech/Project/Results">Our Results</a> |
- | + | ||
- | + | ||
<br><br> | <br><br> | ||
<a href = "https://2014.igem.org/Team:Caltech/Project/Conclusions">Conclusions</a> | <a href = "https://2014.igem.org/Team:Caltech/Project/Conclusions">Conclusions</a> | ||
Line 98: | Line 96: | ||
</td> | </td> | ||
<td valign="top"> | <td valign="top"> | ||
- | <b>For each 25 | + | <b>For each 25 μL reaction mixture: </b> |
- | <ul><li> 12.5 | + | <ul><li> 12.5 μL Phusion Mastermix</li> |
- | <li> 2.5 | + | <li> 2.5 μL primer mix (10 μM of forward and reverse primer)</li> |
- | <li> 1 | + | <li> 1 μL DNA template </li> |
- | <li> 0.75 | + | <li> 0.75 μL DMSO (optional) </li> |
- | <li> Fill to 25 | + | <li> Fill to 25 μL with MilliQ water </li> |
</ul> | </ul> | ||
<b>Thermal Cycler Protocol</b> | <b>Thermal Cycler Protocol</b> | ||
- | <ul><li> | + | <ul><li> 98°C for 30 seconds </li> |
- | <li> | + | <li> 98°C for 10 seconds </li> |
- | <li> | + | <li> 53°C for 30 seconds </li> |
- | <li> | + | <li> 72°C for 15<i>x</i> seconds, where <i>x</i> is the expected length of longest PCR product (in kilobases) </li> |
<li> Repeat above steps 29 more times </li> | <li> Repeat above steps 29 more times </li> | ||
- | <li> | + | <li> 72°C for 10 minutes </li> |
- | <li> Hold at 4C </li> | + | <li> Hold at 4C° </li> |
</ul> | </ul> | ||
Line 128: | Line 126: | ||
<ul><li> Make 1% agarose solution in 1x TBE buffer (typically 0.5 g agarose per 50 mL 1x TBE buffer)</li> | <ul><li> Make 1% agarose solution in 1x TBE buffer (typically 0.5 g agarose per 50 mL 1x TBE buffer)</li> | ||
<li> Microwave solution for 60-90 seconds, until agarose is completely dissolved</li> | <li> Microwave solution for 60-90 seconds, until agarose is completely dissolved</li> | ||
- | <li> Add 5 | + | <li> Add 5 μL SYBR Safe per every 50 μL of agarose solution</li> |
<li> Pour into gel casket and add comb. Let cool for around 20-30 minutes to allow gel to set</li> | <li> Pour into gel casket and add comb. Let cool for around 20-30 minutes to allow gel to set</li> | ||
</ul> | </ul> | ||
<b> Lane mixtures </b> | <b> Lane mixtures </b> | ||
- | <ul><li> For DNA ladders, mix 0.5 | + | <ul><li> For DNA ladders, mix 0.5 μL of ladder, 1 μL loading dye, 4.5 μL MilliQ water </li> |
- | <li> For DNA samples (typically PCR products), mix 2 | + | <li> For DNA samples (typically PCR products), mix 2 μL of sample, 1 μL loading dye, 3 μL MilliQ water </li> |
</ul> | </ul> | ||
<b> Running the gel </b> | <b> Running the gel </b> | ||
Line 151: | Line 149: | ||
<td valign="top"> | <td valign="top"> | ||
<b> If using colonies grown on plates: </b> | <b> If using colonies grown on plates: </b> | ||
- | <ul><li>Pick colonies with pipette tip and re-suspend in 10 | + | <ul><li>Pick colonies with pipette tip and re-suspend in 10 μL of MilliQ water</li> |
</ul> | </ul> | ||
<b> If using liquid cultures: </b> | <b> If using liquid cultures: </b> | ||
- | <ul><li>Add 0.5 | + | <ul><li>Add 0.5 μL of 5mL liquid culture to 10 μL of MilliQ water </li> |
</ul> | </ul> | ||
<b> PCR reaction mixture </b> | <b> PCR reaction mixture </b> | ||
- | <ul><li> 5 | + | <ul><li> 5 μL Phusion Mastermix </li> |
- | <li> 0.5 | + | <li> 0.5 μL forward primer (10 μM)</li> |
- | <li> 0.5 | + | <li> 0.5 μL reverse primer (10 μM)</li> |
- | <li> 4 | + | <li> 4 μL MilliQ water</li> |
- | <li> Make 10<i>x</i> of this reaction mixture, where <i>x</i> is the number of colonies picked </li> | + | <li> Make 10<i>x</i> μL of this reaction mixture, where <i>x</i> is the number of colonies picked </li> |
- | <li> For each colony suspension, add 1 | + | <li> For each colony suspension, add 1 μL of the suspension to 10 μL of the PCR reaction mix </li> |
</ul> | </ul> | ||
<b> Thermal Cycler Protocol </b> | <b> Thermal Cycler Protocol </b> | ||
- | <ul><li> Lid temperature | + | <ul><li> Lid temperature 105°C </li> |
- | <li> | + | <li> 98°C for 10 minutes </li> |
- | <li> | + | <li> 98°C for 30 seconds </li> |
- | <li> | + | <li> 53°C for 15 seconds </li> |
- | <li> | + | <li> 72°C for 15<i>x</i> seconds, where <i>x</i> is the expected length of PCR product (in kilobases) </li> |
<li> Repeat above steps 29 more times </li> | <li> Repeat above steps 29 more times </li> | ||
- | <li> | + | <li> 72°C for 5 minutes </li> |
- | <li> Hold at | + | <li> Hold at 4°C </li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 184: | Line 182: | ||
</td> | </td> | ||
<td valign="top"> | <td valign="top"> | ||
- | <ul><li> Calculate equimolar amounts of each DNA fragment to be used in Gibson assembly. The length of the insert divided by length of the backbone multiplied by the mass of the backbone used (typically 0.5 or 1 ng) gives the mass of insert desired (in ng). Use the concentration of the DNA fragment to determine volume of DNA fragment to be added to the DNA mix. Fill to 2.5 or 5 | + | <ul><li> Calculate equimolar amounts of each DNA fragment to be used in Gibson assembly. The length of the insert divided by length of the backbone multiplied by the mass of the backbone used (typically 0.5 or 1 ng) gives the mass of insert desired (in ng). Use the concentration of the DNA fragment to determine volume of DNA fragment to be added to the DNA mix. Fill to 2.5 or 5 μL of total DNA mix using MilliQ water</li> |
<li> Add 3 times as much Gibson Mastermix as there is DNA mix</li> | <li> Add 3 times as much Gibson Mastermix as there is DNA mix</li> | ||
</ul> | </ul> | ||
<b> Thermal Cycler Protocol </b> | <b> Thermal Cycler Protocol </b> | ||
<ul><li> Lid temperature 105C</li> | <ul><li> Lid temperature 105C</li> | ||
- | <li> | + | <li> 50°C for 1 hour</li> |
- | <li> Hold at | + | <li> Hold at 4°C </li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 203: | Line 201: | ||
<td valign="top"> | <td valign="top"> | ||
<b> For JM109 cells: </b> | <b> For JM109 cells: </b> | ||
- | <ul><li> Add 2 | + | <ul><li> Add 2 μL of plasmid to 20 μL of cells</li> |
<li> Stir with pipette tip</li> | <li> Stir with pipette tip</li> | ||
- | <li> Keep cells on ice</li> | + | <li> Keep cells on ice for 5-10 minutes</li> |
+ | <li> Add 4 volumes of SOC medium and incubate in shaker at 37°C for 1 hour. This step is optional if plating onto CARB. </li> | ||
<li> Plate with glass beads </li> | <li> Plate with glass beads </li> | ||
</ul> | </ul> | ||
- | <b> For DH5&alpha Z1 cells:</b> | + | <b> For DH5α-Z1 cells:</b> |
- | <ul><li> Add 1 | + | <ul><li> Add 1 μL of plasmid to 40 μL of cells </li> |
<li> Incubate on ice for 10 minutes </li> | <li> Incubate on ice for 10 minutes </li> | ||
- | <li> Heat shock at | + | <li> Heat shock at 42°C for 90 seconds </li> |
- | <li> Add 100 | + | <li> Add 100 μL of SOC medium, then incubate in a 37°C shaker for 30-90 minutes to recover </li> |
<li> Plate with glass beads </li> | <li> Plate with glass beads </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr><td bgColor="#777777" colspan="2" height="1px"></td></tr> | ||
+ | |||
+ | <tr> | ||
+ | <td width="25%" valign="top"> | ||
+ | <h4>Liquid Culture Inoculation</h4> | ||
+ | </td> | ||
+ | <td valign="top"> | ||
+ | <ul><li> Add 5 μL antibiotic to 5 mL LB media </li> | ||
+ | <li> Pick colony from solid culture with pipette tip and mix into liquid </li> | ||
+ | <li> Incubate in 37°C shaker overnight</li> | ||
</ul> | </ul> | ||
</td> | </td> | ||
Line 228: | Line 241: | ||
<ul><li> Used to purify PCR products of unwanted DNA </li> | <ul><li> Used to purify PCR products of unwanted DNA </li> | ||
<li> Use QIAquick PCR Purification Kit (Cat. No. 28107)</li> | <li> Use QIAquick PCR Purification Kit (Cat. No. 28107)</li> | ||
- | <li> Deviation from established protocol: elute in 40 | + | <li> Deviation from established protocol: elute in 40 μL EB buffer</li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 242: | Line 255: | ||
<ul><li> Use to extract plasmids from transformed colonies </li> | <ul><li> Use to extract plasmids from transformed colonies </li> | ||
<li> Use QIAprep Spin Miniprep Kit (Cat. No. 27104)</li> | <li> Use QIAprep Spin Miniprep Kit (Cat. No. 27104)</li> | ||
- | <li> Deviation from established protocol: elute in 40 | + | <li> Deviation from established protocol: elute in 40 μL EB buffer</li> |
</ul> | </ul> | ||
</td> | </td> |
Latest revision as of 21:43, 17 October 2014
![]() | |||||||||||||||||||||||||||||||
| |||||||||||||||||||||||||||||||
| |||||||||||||||||||||||||||||||
Project Overview
Project Details Materials and Methods The Experiments Our Results Conclusions References |
|