Team:UC Davis/Protocols

From 2013.igem.org

(Difference between revisions)
 
(5 intermediate revisions not shown)
Line 377: Line 377:
     <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>
Line 383: Line 384:
<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>
Line 391: Line 418:
<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>
Line 402: Line 443:
     <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>
Line 419: Line 460:
<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.   
Line 428: Line 469:
     <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>
Line 446: Line 487:
         <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>
Line 467: Line 509:
         <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">
<table class="gray">
Line 500: Line 542:
<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>
Line 510: Line 552:
     <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 520: 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