Team:UC Davis/Notebook/Protocols
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- | <li ><a href="https://2012.igem.org/" title="Back to iGEM">iGEM</a> | + | <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a> |
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- | <li><a href="https://2012.igem.org/">Main iGEM</a></li> | + | <li><a target="new" href="https://2012.igem.org/">Main iGEM</a></li> |
<li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li> | <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li> | ||
<li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li> | <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li> | ||
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<li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol" | <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol" | ||
title="Data">Ethylene Glycol</a></li> | title="Data">Ethylene Glycol</a></li> | ||
+ | <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Modeling" | ||
+ | title="Data">Modeling</a></li> | ||
+ | |||
<li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li> | <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li> | ||
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+ | <p class="menu_head">Construction of a Transposase Illumina Sequencing Library</p> | ||
+ | <div class="menu_body"> | ||
+ | |||
+ | <p>Procedure</p> | ||
+ | <ul> | ||
+ | <li>Extract genomic DNA using any proprietary gDNA extraction kit. Once DNA is extracted, quantify your DNA and run it on an agarose gel to check DNA quality.</li> | ||
+ | <li>Prepare Illumina Adapters for Transposase Priming, Mix the Following: | ||
+ | <ul><li>10µM No_PCR_Adapter_1: 10µL</li> | ||
+ | <li>10µM No_PCR_Adapter_2: 10µL</li> | ||
+ | <li>10µM Mosaic Element: 20µL</li> | ||
+ | <li>5M NaCl: 1.2µL</li> | ||
+ | <li>Incubate at 98C for 5 mins and let cool to room temperature</li></ul></li> | ||
+ | <li>Prime Transposase | ||
+ | <ul><li>Transposase: 10µL</li> | ||
+ | <li>Annealed adapter (above): 5µL</li> | ||
+ | <li>Water: 15µL</li> | ||
+ | <li>Incubate at room temperature for 20-30 mins.</li></ul></li> | ||
+ | <li>Tagmentation Reaction | ||
+ | <ul><li>Primed Transposase: 10µL (depends on the concentration of available Tn5)</li> | ||
+ | <li>200ng of gDNA extration: XµL</li> | ||
+ | <li>5x Tn5 Buffer: 3µL</li> | ||
+ | <li>Water: up to 15µL</li> | ||
+ | <li>Run multiple reactions in parallel to achieve appropriate amount of input DNA for sequencing platform</li> | ||
+ | <li>Incubate at 55C for 10 mins, and immediately pool reactions and clean up using a PCR clean up kit, elute with 15µL water</li></ul></li> | ||
+ | <li>Gap Filling | ||
+ | <ul><li>Run a typical PCR reaction, without oligos, at 72C for 5 mins</li></ul></li> | ||
+ | <li>Size Selection | ||
+ | <ul><li>Run gap filling products on 2% agarose gel, size select for the appropriate size required for your sequencing platform</li></ul></li> | ||
+ | <li>Quality check your library | ||
+ | <ul><li>Run bioanalyzer to check your library quality, and qPCR to quantify your library before submission.</li></ul></li> | ||
+ | <li>SUBMIT!</li> | ||
+ | </ul> | ||
</div> | </div> | ||
+ | |||
+ | <p class="menu_head">Cutinase Expression and Western Blot</p> | ||
+ | <div class="menu_body"> | ||
+ | |||
+ | <p>Procedure</p> | ||
+ | <ul> | ||
+ | <li>Prepare a starter culture of MG1655 <i>E. coli</i> with pBad regulated and his-tagged LC-Cutinase along with a negative control.</li> | ||
+ | <li>Inoculate new 5 mL cultures from the starter cultures at an OD 600 of about 0.05.</li> | ||
+ | <li>Let the cultures grow until and OD 600 of 0.5, then inoculate two 100 mL cultures (one for the his-tagged protein and one for the negative control). </li> | ||
+ | <li>Once these cultures are at an OD of 0.8, separate into two 50 mL cultures. Induce one at 10 uM, leave the other uninduced.</li> | ||
+ | <li>Take 1 mL samples at different time points.</li> | ||
+ | <li>Centrifuge samples at 5000g for 5 minutes then take off and save media.</li> | ||
+ | <li>Wash cells with water.</li> | ||
+ | <li>Resuspend with 300 uL of B-PER Protein Extraction Reagent and follow the instructions provided with that kit. After this, there should be samples of culture media, and soluble and insoluble whole cell lysate.</li> | ||
+ | <li> Take 15 uL of each sample, including controls, and run on western blot.</li> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <p class="menu_head">pNPB Assay</p> | ||
+ | <div class="menu_body"> | ||
+ | |||
+ | <p>Materials</p> | ||
+ | <ul> | ||
+ | <li>pNPB buffer (should be made and used in fumehood due to acetonitriles toxicity) | ||
+ | <ul><li>10mM pNPB in acetonitrile</li> | ||
+ | <li>1:4:95 ratio of acetonitrile pNPB solution, 100% ethanol and 50mM Tris-HCL (pH 8)</li></ul></li> | ||
+ | <li>LB with specific resistance</li> | ||
+ | <li>Cell Culture</li> | ||
+ | <li>96 well plate</li></ul> | ||
+ | <br> | ||
+ | <p>Protocol</p> | ||
+ | <ul><li>First assign each well of the plate except for outer wells which should be filled with LB to prevent evaporation of medium.</li> | ||
+ | <li>Fill wells with 95 uL of LB</li> | ||
+ | <li>Fill wells with 5 uL of cell culture</li> | ||
+ | <li>In fumehood, add 100 uL of bufer to each well</li> | ||
+ | <li>Run in Tecan taking both ODs and absorbance 405 readings</li> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <p class="menu_head">SDM (Site Directed Mutagenesis)</p> | ||
+ | <div class="menu_body"> | ||
+ | |||
+ | <p>Materials</p> | ||
+ | <ul> | ||
+ | <li>Pfu turbo</li> | ||
+ | <li>10X Pfu turbo buffer</li> | ||
+ | <li>dNTPs (10mM)</li> | ||
+ | <li>Forward and reverse primers (0.1ug/uL, see methods section for design tips)</li> | ||
+ | <li>dH<sub>2</sub>0</li> | ||
+ | <li>Dpn1</li> | ||
+ | <li>Competent cells</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <p>Methods</p> | ||
+ | <ul> | ||
+ | <li>Primer Design</li> | ||
+ | <ul> | ||
+ | <li>Forward primer should be between 25 and 45 bases in length and contain the desired mutation in the center with correct sequences on both sides; the reverse primer is the reverse complement of this.</li> | ||
+ | <li>Primers should have a minimum GC content of 40% and terminate in one or more C's or G's.</li> | ||
+ | <li>Tm should be greater or equal to 78°C and can be calculated as follows:</li> | ||
+ | <li>Tm = 81.5 + 0.41(%GC) - 675/(length in bases) - %mismatch</li> | ||
+ | </ul></ul> | ||
+ | <ul> | ||
+ | <li>Reaction</li> | ||
+ | <ul> | ||
+ | <li>Template DNA: 1 µL</li> | ||
+ | <li>10X Buffer: 5 µL</li> | ||
+ | <li>Forward Primer (100 ng/µL): 1µL</li> | ||
+ | <li>Reverse Primer (100 ng/µL): 1µL</li> | ||
+ | <li>10mM dNTPs: 1µL</li> | ||
+ | <li>Pfu Turbo (Stratagene): 1µL</li> | ||
+ | <li>MilliQ H<sub>2</sub>0: 40µL</li> | ||
+ | </ul> | ||
+ | <li>PCR Program</li> | ||
+ | <ul> | ||
+ | <li>95°C for 1 minute</li> | ||
+ | <li>95°C for 50 seconds, 60°C for 50 seconds, 68°C for 1 minute/kb of plasmid length -- repeat this step 17 times, or 18 cycles total</li> | ||
+ | <li>68°C for 7 minutes</li> | ||
+ | <li>4°C hold</li> | ||
+ | <li>Following PCR - save 4µL of PCR reaction.</li> | ||
+ | <li>To remaining 46µLs add 1µL of Dpn1 to PCR reaction (PCR cleanup is not necessary). Incubate at 37°C for 1-2 hours to digest parental DNA. Run 5µL of the digested reaction on a gel and compare to the undigested parental plasmid - there should be some difference in band pattern. Transform into competent cells (PCR cleanup is not necessary).</li> | ||
+ | </ul> | ||
+ | |||
+ | <br> | ||
+ | <li>Protocol Adapted From <a href="http://mcmanuslab.ucsf.edu/protocol/site-directed-mutagenesis-stratagene-protocol">Here</a> | ||
+ | </ul> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
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href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol "> | href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol "> | ||
Ethylene Glycol</a> </li><li><a style="color:#000000 " | Ethylene Glycol</a> </li><li><a style="color:#000000 " | ||
+ | href="https://2012.igem.org/Team:UC_Davis/Data/Modeling "> | ||
+ | Modeling</a> </li><li><a style="color:#000000 " | ||
+ | |||
href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul> | href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul> | ||
Latest revision as of 02:52, 27 October 2012