Team:UC Davis/Protocols

From 2013.igem.org

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Line 192: Line 192:
<li>10 g      NaCl</li>
<li>10 g      NaCl</li>
<li>5 g        Yeast Extract</li>
<li>5 g        Yeast Extract</li>
-
1 L  Total
+
1 L  Total (add dH<sub>2</sub>0 to reach total volume)
<li>Add 15 g Agar, if being poured into plates.</li>
<li>Add 15 g Agar, if being poured into plates.</li>
<li>Autoclave, when cool add antibiotics if desired.</li>
<li>Autoclave, when cool add antibiotics if desired.</li>
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<li>Preheat water bath to 42º C.</li>
<li>Preheat water bath to 42º C.</li>
<li>Thaw competent cells on ice for 10 minutes.</li>
<li>Thaw competent cells on ice for 10 minutes.</li>
-
<li>Use 50 µL competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (PUC19 carb resistance). Swirl to mix, store on ice 5 minutes. </li>
+
<li>Use 50 µL chemically competent cells, add transforming DNA [up to 25 ng per 50 µL of cells, volume not exceeding 2.5 µL (5%)]. For control add 1 µL of control DNA (e.g. PUC19). Swirl gently to mix, store on ice 5 minutes. </li>
<li>Heat shock in 42º C water bath for 45 seconds.</li>
<li>Heat shock in 42º C water bath for 45 seconds.</li>
-
<li>Cool cells in ice bath for a few minutes.</li>
+
<li>Cool cells in ice bath for 1-2 minutes.</li>
<li>Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes. </li>
<li>Add 800 µL LB to each tube, set in shaker at 37º C for 45 minutes. </li>
-
<li>Transfer 200 µL of culture per plate (containing the appropriate antibiotic).</li>
+
<li>Transfer 200 µL of culture to a plate (containing the appropriate antibiotic).</li>
<li>Spread using glass beads.</li>
<li>Spread using glass beads.</li>
<li>Invert plates and incubate overnight (12-16 hrs) at 37º C.</li>
<li>Invert plates and incubate overnight (12-16 hrs) at 37º C.</li>
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<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li>
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li>
-
50 µL  Total                      → 37º C, 3 hrs.
+
50 µL  Total                      → incubate at 37º C for 3 hrs.
<p>Procedure</p>
<p>Procedure</p>
<li>Treat insert with XbaI and PstI</li>
<li>Treat insert with XbaI and PstI</li>
<li>Treat vector with SpeI and PstI</li>
<li>Treat vector with SpeI and PstI</li>
 +
<li>After incubating at 37º C for 3 hrs, either heat inactivate the enzymes or proceed directly to gel extraction and purification.</li>
     </div>
     </div>
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<li>2 µL T4 10x Buffer</li>
<li>2 µL T4 10x Buffer</li>
<li>1 µL DNA ligase</li>
<li>1 µL DNA ligase</li>
-
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li>
+
<li>Add appropriate amount of dH<sub>2</sub>0 (µL) to reach total volume</li>
20 µL Total            →Leave at room temperature for 20 minutes
20 µL Total            →Leave at room temperature for 20 minutes
<br></br>
<br></br>
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<li>2 µL Buffer</li>
<li>2 µL Buffer</li>
<li>1 µL ligase</li>
<li>1 µL ligase</li>
-
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li>
+
<li>Add appropriate amount of dH<sub>2</sub>0 (µL) to reach total volume</li>
20 µL total  
20 µL total  
<br></br>
<br></br>
Line 312: Line 313:
<li>2 µL Buffer</li>
<li>2 µL Buffer</li>
<li>1 µL ligase</li>
<li>1 µL ligase</li>
-
<li>Add appropriate amount of dH<sub>2</sub>0 (µL)</li>
+
<li>Add appropriate amount of dH<sub>2</sub>0 (µL) to reach total volume</li>
20 uL Total
20 uL Total
<p>Procedure</p>
<p>Procedure</p>
-
<li>Mix these materials in the amounts determined by the reaction volume calculator <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.
+
<li>Mix these materials in the amounts determined by the reaction volume calculator for the vector and insert DNA <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.
     </div>
     </div>
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<p>Procedure</p>
<p>Procedure</p>
<ol>
<ol>
-
<li>Add 0.5 grams of agarose to 50 mL of 1X TAE buffer.</li>
+
<li>Add 0.5 grams of agarose to 50 mL of 1X TAE buffer. (1% agarose gel)</li>
-
<li>Microwave agarose/TAE until agarose completely dissolved.</li>
+
<li>Microwave agarose/TAE until agarose is completely dissolved. (Caution: Will be hot)</li>
-
<li>Cool under water, add SYBR safe dye (2.5-3µL).</li>
+
<li>Cool under water or let sit until cool.</li>
 +
<li>Add SYBR safe dye (2.5-3µL).</li>
<li>Pour into mold with appropriate comb.</li>
<li>Pour into mold with appropriate comb.</li>
<li>Wait 15 minutes for gel to solidify.</li>
<li>Wait 15 minutes for gel to solidify.</li>
-
<li>Load DNA with dye into wells while submerged in 1X TAE.</li>
+
<li>Transfer gel to a gel electrophoresis box.</li>
 +
<li>Load DNA and appropriate DNA standard or ladder with dye into wells while submerged<br /> in 1X TAE buffer.</li>
<li>Run gel at a constant 120V.</li>
<li>Run gel at a constant 120V.</li>
-
<li>Check gel periodically.</li>
+
<li>Check gel periodically until the dye has run the desired length of the gel.</li>
 +
<li>View gel under short wavelength or UV light, with proper protection, to check for bands.</li>
</ol>
</ol>
</td>
</td>
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<p>Procedure</p>
<p>Procedure</p>
<ol>
<ol>
-
<li>Prepare agarose gel and use 3 combs to make a bigger well.</li>
+
<li>Prepare agarose gel (see gel electrophoresis) and use additional or larger combs to make larger wells.</li>
-
<li>Once it has run, use hand held UV lamp (in the dark, wearing goggles) to identify bands. </li>
+
<li>Once the gel has run, use preferably a blue light lamp, or UV for a very short time (it will degrade your DNA), (in the dark, wearing goggles and skin protection) to identify bands. </li>
-
<li>Cut out desired band with stamp pipette tip and transfer to a clean tube.  The stamp pipette tip can be left in the tube to be cleaned out with a smaller pipette tip. </li>
+
<li>Cut out the desired bands with a stamp pipette tip or clean razor and transfer to a clean tube.  The stamp pipette tip can be left in the tube to be cleaned out with a smaller pipette tip. </li>
 +
<table class="gray">
 +
<tr>
 +
<td>
<li>Weigh gel fragments and add 200 µL Buffer NTI for every 100 mg agarose gel.</li>
<li>Weigh gel fragments and add 200 µL Buffer NTI for every 100 mg agarose gel.</li>
  Incubate sample for 5-10 min at 50º C, vortexing every 2-3 min until the gel is completely dissolved.</li>
  Incubate sample for 5-10 min at 50º C, vortexing every 2-3 min until the gel is completely dissolved.</li>
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<li>Centrifuge for 1 min at 11,000 x g. </li>
<li>Centrifuge for 1 min at 11,000 x g. </li>
</ol>
</ol>
 +
</td>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/e/ec/UCDavis_gelExt.jpg" width=300 height=183 />
 +
</td>
 +
</tr>
 +
</table>
     </div>  
     </div>  
-
   <p class="menu_head">PCR Amplification for Golden Gate Assembly</p>
+
   <p class="menu_head">PCR Amplification</p>
     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
 +
<li>10 µL 5x HF Buffer</li>
<li>10 µL 5x HF Buffer</li>
<li>1  uL dNTPs</li>
<li>1  uL dNTPs</li>
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<li>100 ng Template DNA (2 ng/µL)</li>
<li>100 ng Template DNA (2 ng/µL)</li>
<li>0.5 µL DNA Phusion Polymerase</li>
<li>0.5 µL DNA Phusion Polymerase</li>
-
<li>Add appropriate amount of ddH<sub>2</sub>O</li>
+
<li>Add appropriate amount of ddH<sub>2</sub>O to reach total volume</li>
-
50 µL Total
+
50 µL Total  
-
<li>Run 1% agarose gel for verification. If the gel is good, perform PCR clean up.</li>
+
 +
<p><br />
 +
or for DNA with high G/C content we found a combination of Taq and Pfu gave us better results.
 +
</p>
 +
<li>5 µL 10x Buffer</li>
 +
<li>10 uL Q solution</li>
 +
<li>2.5 µL Forward Primer</li>
 +
<li>2.5 µL Reverse Primer</li>
 +
<li>1.25 µL DNTPs</li>
 +
<li>100 ng Template DNA (2 ng/µL)</li>
 +
<li>0.3 µL Taq DNA Polymerase</li>
 +
<li>0.1 µL Cloned Pfu DNA Polymerase</li>
 +
<li>Add appropriate amount of ddH<sub>2</sub>O to reach total volume</li>
 +
50 µL Total
 +
<p><br />
 +
PCR program
 +
<ol>
 +
<li>98º C 30 sec</li>
 +
<li>98º C 10 sec</li>
 +
<li>___º C 30 sec  (Temperature depends on Tm of your primers)</li>
 +
<li>72º C 1 min / kb Repeat Steps 2-4 29x (30x total)</li>
 +
<li>72º C 5 min</li>
 +
<li>4º C Hold</li>
 +
</ol>
 +
</p>
 +
 
 +
<li>Run PCR products on a 1% agarose gel for verification. If the gel is good, perform PCR clean up with your kit of choice.</li>
</div>
</div>
<p class="menu_head">Golden Gate Assembly</p>
<p class="menu_head">Golden Gate Assembly</p>
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<p>Materials</p>
<p>Materials</p>
-
<li>100 ng for each DNA part</li>
+
<li>40 fmol of DNA for each part (or 100 ng if your parts are all roughly the same size)</li>
-
<li>1 µL BsaI</li>
+
<li>.75 µL BsaI</li>
-
<li>1 µL T4-ligase</li>
+
<li>.2 µL 100x BSA</li>
 +
<li>1 µL T4 DNA ligase</li>
<li>2 µL 10X T4 ligase buffer</li>
<li>2 µL 10X T4 ligase buffer</li>
-
<li>Add appropriate amount of ddH<sub>2</sub>0.</li>
+
<li>Add appropriate amount of ddH<sub>2</sub>0 to reach total volume.</li>
20 µL Total
20 µL Total
 +
<p><br />
 +
To convert your DNA concentration to fmol/µL, use the equation 1µg of 1kb DNA = 1.52 pmol.
 +
<br /><br />
 +
</p>
 +
<p>PCR Program</p>
 +
<ol>
 +
<li>37º C 2 min</li>
 +
<li>16º C 3 min Repeat Steps 1-2 49x (50x total)</li>
 +
<li>50º C 5 min</li>
 +
<li>80º C 5 min</li>
 +
<li>4º C Hold</li>
 +
</ol>
 +
<li>Following PCR, directly transform 5-10 µL of your product into competent cells.</li>
</div>
</div>
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     <div class="menu_body">
     <div class="menu_body">
-
<p>Procedure</p>
+
<p>Procedure (Invitrogen Quick Plasmid Miniprep Kit)</p>
<ol>
<ol>
<li>Sediment the cells by centrifuging 1-5 mL of overnight LB-culture. Remove all medium.</li>
<li>Sediment the cells by centrifuging 1-5 mL of overnight LB-culture. Remove all medium.</li>
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<p>Procedure</p>
<p>Procedure</p>
-
<li>Add equal volumes (500-700 µL) of overnight cell culture and glycerol into a cryotube, keep sterile with a flame.</li>
+
<li>Add equal volumes (500-700 µL) of overnight cell culture and 80% glycerol into a cryotube, keep sterile with a flame.</li>
<li>Store at -80º C.</li>
<li>Store at -80º C.</li>
<li>When reviving a glycerol stock, keep the glycerol stock on dry ice.   
<li>When reviving a glycerol stock, keep the glycerol stock on dry ice.   
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     <div class="menu_body">
     <div class="menu_body">
-
<p>Procedure</p>
+
<p>Procedure (Quintara Biosciences)</p>
-
<li>Primer will have [ng] content printed on label: add 10x H<sub>2</sub>0 for DNA at 100 uM.</li>
+
<li>Primer will have [ng] content printed on label: add 10x H<sub>2</sub>0 for DNA at 100 µM.</li>
-
<li>Need 10 uM for sequencing, so dilute a portion of the hydrated primer solution 10x.</li>
+
<li>Need 10 µM solution for sequencing, so dilute a portion of the hydrated primer solution 10x (e.g. 1 µL primer + 9 µL H<sub>2</sub>0).</li>
<li>Determine DNA concentration of template DNA.</li>
<li>Determine DNA concentration of template DNA.</li>
<li>(Premixed) in 0.5 µL tube</li>
<li>(Premixed) in 0.5 µL tube</li>
<li>.6 µg    DNA (final concentration: 50 ng/µL)</li>
<li>.6 µg    DNA (final concentration: 50 ng/µL)</li>
-
<li>8 pmol primer (universal primers 10 µM = .8 µL)</li>
+
<li>8 pmol primer (10 µM = .8 µL)</li>
-
<li>Add appropriate amount of H<sub>2</sub>O.</li>
+
<li>Add appropriate amount of H<sub>2</sub>O for total volume.</li>
12 µL total
12 µL total
     </div>
     </div>
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         <p>Procedure</p>
         <p>Procedure</p>
<ol>
<ol>
-
<li>Log in to nanodrop program.</li>
+
<li>Log in to nanodrop program and select Nucleic Acids/DNA.</li>
-
<li>Moisten a Kim wipe and clean the pedestal.</li>
+
<li>Moisten a Kimwipe and clean the pedestal.</li>
<li>Apply 2 µL H<sub>2</sub>O to pedestal and click 'OK'.</li>
<li>Apply 2 µL H<sub>2</sub>O to pedestal and click 'OK'.</li>
<li>Press 'Blank' button.</li>
<li>Press 'Blank' button.</li>
-
<li>Wipe blank from pedestal using Kimwipe.</li>
+
<li>Gently wipe blank from pedestal using a Kimwipe.</li>
<li>Apply 2 µL of desired sample to pedestal.</li>
<li>Apply 2 µL of desired sample to pedestal.</li>
<li>Click 'Measure'.</li>
<li>Click 'Measure'.</li>
-
<li>Print results. </li>
+
<li>Gently wipe pedestal using a Kimwipe and continue measuring remaining samples.</li>
 +
<li>Record/print results. </li>
</ol>
</ol>
</td>
</td>
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         <p>Procedure</p>
         <p>Procedure</p>
<ol>
<ol>
-
<li>Grow cultures overnight in LB at 37 C, 150 RPM. </li>
+
<li>Grow cultures overnight, or until OD<sub>600</sub>>1, in LB at 37 C, 150 RPM. </li>
<li>Measure OD<sub>600</sub> and dilute to get <0.01 OD<sub>600</sub>.</li>
<li>Measure OD<sub>600</sub> and dilute to get <0.01 OD<sub>600</sub>.</li>
<li>Grow until the OD<sub>600</sub> approaches 0.5.</li>
<li>Grow until the OD<sub>600</sub> approaches 0.5.</li>
-
<li>Load 96 well plate with LB or M9, depending on the experiment, as well as the appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD<sub>600</sub> of 0.1 in 200 µL. </li>
+
<li>Load 96 well plate with LB, or other media of choice, depending on the experiment, as well as the appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD<sub>600</sub> of 0.1 in 200 µL. If changing media, first spin down the culture to pellet, decant and aspirate any remaining media, and resuspend in new media to reach the desired OD<sub>600</sub> before loading in plate.</li>
-
<li>Be sure to include the appropriate positive and negative controls.</li>
+
<li>Be sure to include the appropriate positive and negative controls (e.g. blank media, strain control, etc.).</li>
</ol>
</ol>
 +
<table class="gray">
 +
<tr>
 +
<td>
<p>Tecan Program Parameters</p>
<p>Tecan Program Parameters</p>
<ul>
<ul>
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<li>Gain: 35</li>
<li>Gain: 35</li>
</ul>
</ul>
 +
</td>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/5/54/UCDavis_tecantest.jpg" width=403 height=200 />
 +
</td>
 +
</tr>
 +
</table>
</div>
</div>
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<li> Identify site that needs to be mutated.</li>
<li> Identify site that needs to be mutated.</li>
<li>Check the amino acid sequence to create a silent mutation, generally the last base in a codon.</li>
<li>Check the amino acid sequence to create a silent mutation, generally the last base in a codon.</li>
-
<li>Check a codon usage table to help choose how the codon should be changed, try to pick a frequently used codon. </li>
+
<li>Check a codon usage table for your chassis to help choose how the codon should be changed, try to pick a frequently used codon. </li>
-
<li>Take about 20 base pairs upstream and 20 base pairs downstream of your desired mutation site to create your primer, try to have it start and end in a G or C. The sequence should be identical to the template except for the one changed base you are trying to mutate at the center.  </li>
+
<li>Take about 20 base pairs upstream and 20 base pairs downstream of your desired mutation site to create your primer. The sequence should be identical to the template except for the one changed base you are trying to mutate at the center.  </li>
<li>The reverse primer will be the reverse complement of this sequence.</li>
<li>The reverse primer will be the reverse complement of this sequence.</li>
</ol>
</ol>
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     <div class="menu_body">
     <div class="menu_body">
                 <p>Materials</p>
                 <p>Materials</p>
-
<li>Primer will have [µg] content printed on label: add H<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li>
+
<li>Primer will have [µg] content printed on label: add dH<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li>
-
<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x.</li>
+
<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x (e.g. 1 µL primer + 9 µL dH<sub>2</sub>0).</li>
<li>Determine DNA concentration of template DNA.</li>
<li>Determine DNA concentration of template DNA.</li>
Line 498: Line 562:
<li>1 µL      dNTPs (10 mM)</li>
<li>1 µL      dNTPs (10 mM)</li>
<li>1 µL      Pfu Turbo (enzyme)</li>
<li>1 µL      Pfu Turbo (enzyme)</li>
-
<li>Add appropriate amount of dH<sub>2</sub>O.</li>
+
<li>Add appropriate amount of dH<sub>2</sub>O to reach total volume.</li>
50 µL Total
50 µL Total
<br></br>
<br></br>

Latest revision as of 22:26, 19 October 2013

Protocols