From 2012.igem.org

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Protocols

Registry Part Distribution Rehydration

Add 20 µL sterile H2O to desired well in distribution plate.
Incubate at room temperature for ~10 minutes.
Transfer resuspension to microcentrifuge tube.

Transformations

Materials

Competent Cells
DNA template
800 µL LB
LB+Antibiotic Plates

Procedure

Thaw competent cells on ice.
Transfer 50 µL of competent cells to chilled falcon tubes.
Add 1 µL of template to cells (2.5 µL if dilute).
Incubate on ice for 30 minutes.
Heat schock in 42 °C water bath for 90 seconds.
Immediately place back onto ice for 2 minutes.
Add 800 µL of LB to each tube.
Incubate at 37 °C for 1 hour.
Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.
Incubate overnight at 37 °C.

Restriction Enzyme Double Digest

Materials

Procedure

Mix reactants, adding enzyme last.
Place at 37 °C for 3-5 hours.
If not purifying or running on a gel immediately, increase to 80 °C for 20 minutes to kill enzymes (some enzymes need only a 65 °C heatkill, check enzyme).
Run on a gel and extract product.

PCR (sequence dependent)

Materials

Procedure

Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
Run in thermal cycler.

PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension)

Materials

10 µL Q Solution
5 µL 10x Buffer
1.25 µL 10mM dNTPs
2.5 µL Forward Primer
2.5 µL Reverse Primer
0.3 µL Taq
0.15 µL PFU
Template based on concentrations determined by SOEing calculator: “Link”
±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”

Procedure

Mix reactants into PCR tubes.
Run in thermal cycler.
Continue PCR SOEing of parts until completed.

Ligation

Materials

Digested Vector
Digested Insert
Water
T4 DNA Ligase
T4 DNA Ligase Buffer

Procedure

Mix these materials in the amounts determined by the reaction volume calculator here.

Ethylene Glycol Media

Materials

34 mM NaH₂PO₄
64 mM K₂HPO₄
20 mM (NH₄)₂SO₄
1 µM FeSO₄
0.1 mM MgSO₄
10 µM CaCl₂
30 mM Ethylene Glycol
30 mM Glycolate
0.5% wt/vol Casein Acid Hydrolysate
1.5% wt/vol Agar (if making solid media)

Procedure

Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.
Mix together NaH₂PO₄, K₂HPO₄, (NH₄)₂SO₄, FeSO₄, MgSO₄, and CaCl₂.
Titrate to pH 7 with HCl.
Add the glycolate and casein acid hydrolysate.
Mix in the ethylene glycol in a fume hood.
If making solid media, also mix in agar.
Autoclave on an appropriate cycle.

Ethylene Glycol Toxicity Assay

Materials

Luria Broth Media
Ethylene Glycol
Tecan M200 Pro
E. coli

Procedure

Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.
Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
Incubate at 37°C overnight on a shaker at 150 rpm.
From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.
Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.
Add “blank” wells of just LB media to an entire column to serve as a control for the LB.
Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.
Aliquot the remaining dilutions as the diagram below depicts.
Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.
Follow the steps of your Tecan machine appropriate to the specific bacterial strain.

EMS (Ethyl methanesulfonate Mutagenesis)

Materials

Buffer A: Minimal medium + 0.2 M Tris (pH 7.5). per liter:
- K₂HPO₄: 10.5g
- KH₂PO₄: 4.5g
- (NH₄)₂SO₄: 1g
- Sodium Citrate * 2H₂O: 0.5g
- 1M Tris (pH 7.5): 200mL Tris

_{Obtained components from “Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY)” pg 432}

EMS (Sigma)
50mL conical Corning tubes
15mL Falcon tubes
LB Broth
Ethylene Glycol Agar Plates

Procedure

1. Prepare a 10mL liquid culture in LB in a 50mL conical tube and grow it overnight until it reaches OD 0.2.
Chill the cells on ice and spin down the 10mL aliquots at max speed in Eppendorf Centrifuge 5810 R for 10 minutes.
Wash twice with 10mL Buffer A.

Pipet up and down to mix, pellet cells, decant supernatant.

Re-suspend the pellet in 5mL of Buffer A and transfer the medium to a Falcon tube.
Add (35μL, 70μL, or 105μL) of EMS into each tube of re-suspended cells.
Close tubes and parafilm the lid 2X.
Vortex to mix and place in a secondary container (50mL conical tube).
Transfer to mixing platform and agitate at ~30 rpm at 37°C.
Withdraw samples at fixed time points.
At each time point, take 1mL aliquots of the sample and place in a 15mL Falcon tube. Place in a secondary container (50mL Conical tube) and spin at max speed in Eppendorf Centrifuge 5810 R for 5 minutes.
Discard supernatant in waste container.
Wash twice in 5mL of Buffer A and discard supernatant in waste container.
Re-suspend in 5mL of Buffer A and titer for viable cells.
Add 0.5mL of mutagenized culture into 10mL of LB broth in a 50mL conical tube.
Grow cultures overnight at 37°C and plate for mutants on EG agar plates.
Look for viable cells

Safety: Health Hazards

Acute toxicity (oral, dermal, inhalation), category 4
Skin irritation, category 2
Eye irritation, category 2
Skin sensitization, category 1
Specific Target Organ Toxicity – Single exposure, category 3
LD50 – 470mg/kg

Recommended Safety & Handling

Always work in fume hood and wear lab coat, goggles, and gloves.

Construction of a Transposase Illumina Sequencing Library

Procedure

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.
Prepare Illumina Adapters for Transposase Priming, Mix the Following:
- 10µM No_PCR_Adapter_1: 10µL
- 10µM No_PCR_Adapter_2: 10µL
- 10µM Mosaic Element: 20µL
- 5M NaCl: 1.2µL
- Incubate at 98C for 5 mins and let cool to room temperature
Prime Transposase
- Transposase: 10µL
- Annealed adapter (above): 5µL
- Water: 15µL
- Incubate at room temperature for 20-30 mins.
Tagmentation Reaction
- Primed Transposase: 10µL (depends on the concentration of available Tn5)
- 200ng of gDNA extration: XµL
- 5x Tn5 Buffer: 3µL
- Water: up to 15µL
- Run multiple reactions in parallel to achieve appropriate amount of input DNA for sequencing platform
- Incubate at 55C for 10 mins, and immediately pool reactions and clean up using a PCR clean up kit, elute with 15µL water
Gap Filling
- Run a typical PCR reaction, without oligos, at 72C for 5 mins
Size Selection
- Run gap filling products on 2% agarose gel, size select for the appropriate size required for your sequencing platform
Quality check your library
- Run bioanalyzer to check your library quality, and qPCR to quantify your library before submission.
SUBMIT!

Cutinase Expression and Western Blot

Procedure

Prepare a starter culture of MG1655 E. coli with pBad regulated and his-tagged LC-Cutinase along with a negative control.
Inoculate new 5 mL cultures from the starter cultures at an OD 600 of about 0.05.
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).
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.
Take 1 mL samples at different time points.
Centrifuge samples at 5000g for 5 minutes then take off and save media.
Wash cells with water.
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.
Take 15 uL of each sample, including controls, and run on western blot.

pNPB Assay

Materials

pNPB buffer (should be made and used in fumehood due to acetonitriles toxicity)
- 10mM pNPB in acetonitrile
- 1:4:95 ratio of acetonitrile pNPB solution, 100% ethanol and 50mM Tris-HCL (pH 8)
LB with specific resistance
Cell Culture
96 well plate

Protocol

First assign each well of the plate except for outer wells which should be filled with LB to prevent evaporation of medium.
Fill wells with 95 uL of LB
Fill wells with 5 uL of cell culture
In fumehood, add 100 uL of bufer to each well
Run in Tecan taking both ODs and absorbance 405 readings

SDM (Site Directed Mutagenesis)

Materials

Pfu turbo
10X Pfu turbo buffer
dNTPs (10mM)
Forward and reverse primers (0.1ug/uL, see methods section for design tips)
dH₂0
Dpn1
Competent cells

Methods

Primer Design

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.
Primers should have a minimum GC content of 40% and terminate in one or more C's or G's.
Tm should be greater or equal to 78°C and can be calculated as follows:
Tm = 81.5 + 0.41(%GC) - 675/(length in bases) - %mismatch

Reaction

PCR Program

95°C for 1 minute
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
68°C for 7 minutes
4°C hold
Following PCR - save 4µL of PCR reaction.
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).

Protocol Adapted From Here

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+}
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+color: white;
+}
+#myleftbox .threeright
+{
+width:175px;
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+background-color:#ba9108;
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+font-size: 13px;
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+}
+#myleftbox .twoboxes
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+}
+#myleftbox .twoboxes1
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+font: sans-serif;
+font-size: 13px;
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+left:9px;
+}
+#myleftbox .twoboxes:hover {
+background-color:red;
+}
+#myleftbox .twoboxes1:hover {
+background-color:green;
+}
+#sponsorbox
+{
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+width: 208px;
+margin-top:15px;
+float: right;
+height: auto;
+}
+#sponsorbox .sponsorfloat
+{
+left:-3px;
+width:198px;
+float: right;
+background-color: #004b85;
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+border-radius: 4px;
+padding: 5px 5px 5px 5px;
+font: sans-serif;
+font-size: 13px;
+color: rgba(225,225,225,1);
+}
+#sponsorbox .sponsorfloat h2
+{
+#color:#ba9108;
+color:white;
+font-size:130%;
+}
+#sponsorbox .sponsorfloat a
+{
+color:white;
+font: sans-serif;
+}
+#sponsorbox .sponsorfloat p
+{
+font: sans-serif;
+}
+.newsAnnouncement
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 }
@@ Line 287: / Line 801: @@
 font-weight:bold;
 font-size: 40px;
-color: #004b85;;
+color: #016D8B;;
 left:10px;
 }
@@ Line 323: / Line 837: @@
+#firstpane {
+	position:relative;
+}
+#firstpane p {
+display:block;
+}
+#firstpane .menu_head {
+	padding: 5px 20px;
+	cursor: pointer;
+	position: relative;
+	margin:1px;
+       font-weight:bold;
+       background: #eef4d3 url(https://static.igem.org/mediawiki/2012/1/1d/UCD_arrow_right.png) center left no-repeat;
+	background-position: 5px center;
+        background-size: 0.7em auto;
+}
+#firstpane .menu_body {
+	display:none;
+	background-color:#e1e1e1;
+        background-image:none !important;
+        margin-top:-5px
+        border-radius:4px;
+        padding:10px;
+}
+#firstpane .menu_body a {
+  display:block;
+  color:#006699;
+  color:black;
+  background-color:#E1E1E1;
+  padding-left:10px;
+  font-weight:bold;
+  text-decoration:none;
+}
+#firstpane .menu_body a:hover {
+  color: #000000;
+  text-decoration:underline;
+}
+#catlinks {
+background-color:transparent;
+border:1px solid transparent;
+}
+#footpage {
+background-color:transparent;
+}
+#footer-box {
+background-color:rgba(191,191,191,0.5);
+margin-top:0px;
+}
 #footer-box-1 {
@@ Line 331: / Line 895: @@
          border: 1px solid #444444;
 }
+#tweets {
+    width: 203px;
+    margin: 0 auto;
+    font: sans-serif;
+    font-size: 12px;
+}
+#tweets .twtr-widget,
+#tweets .twtr-doc {
+    width: 100%;
+    height: auto;
+}
+#tweets .twtr-hd {
+    display:none;
+    background: #d40;
+    color: #fff;
+    -moz-border-radius: 4px 4px 0 0;
+    border-radius: 4px 4px 0 0;
+    font-family: sans-serif !important;
+    }
+#tweets .twtr-hd *,
+#tweets .twtr-hd h4 a {
+    background: #d40 !important;
+    font: sans-serif !important;
+}
+#tweets .twtr-hd h3,
+#tweets .twtr-hd h4 {
+    font-weight: normal;
+    text-align: left;
+    margin:0;
+}
+#tweets .twtr-hd h3 {
+    background: #fff !important;
+    color: #333 !important;
+    font: sans-serif;
+    font-size: 1.0em !important;
+    padding: 0.2em !important;
+    -moz-border-radius: 0px;
+    border-radius: 0px;
+}
+#tweets .twtr-tweet {
+    background: #fff;
+    font: sans-serif !important;
+    font-size: 12px;
+}
+#tweets .twtr-tweet a:link,
+#tweets .twtr-tweet a:visited,
+#tweets .twtr-tweet a:hover {
+    color: #c40 !important;
+}
+#tweets .twtr-tweet .twtr-tweet-text {
+    border-bottom: 1px dashed #666 !important;
+    padding-bottom: 4px !important;
+    font: sans-serif;
+    font-size:12px;
+}
+#tweets .twtr-tweet .twtr-tweet-text p {
+    color: #333 !important;
+    font: sans-serif;
+    font-size: 12px;
+}
+#overlay {
+  z-index:2;
+  position:fixed;
+  top: 0;
+  left: 0;
+  height: 100%;
+  width: 100%;
+  background: black no-repeat scroll center center;
+}
+#lightbox {
+  position: fixed;
+  width:840;
+  z-index:2;
+}
 </style>
@@ Line 346: / Line 1,006: @@
   	  <ul>
 			   <li style='color:#014457; cursor:default'><a>teams</a></li>
-  	                   <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis">Page</a></li>
+  	                   <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols">Page</a></li>
-                		   <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis&amp;action=edit&amp;redlink=1">Discussion</a></li>
+                		   <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis/Notebook/Protocols&amp;action=edit&amp;redlink=1">Discussion</a></li>
                 <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&action=edit">Edit</a></li>
-                            <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=history">History</a></li>
+                            <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&amp;action=history">History</a></li>
-                		   <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis">Move</a></li>
+                		   <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis/Notebook/Protocols">Move</a></li>
-                            <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=watch">Watch</a></li>
+                            <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&amp;action=watch">Watch</a></li>
 			   <li><a href="https://igem.org/Login">Log in</a></li>
@@ Line 359: / Line 1,019: @@
     <div id="newnavi">
       <ul class="newmenu">
-        <li ><a href="https://2012.igem.org/" title="Back to iGEM">iGEM</a></li>
+     <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
+          <ul>
+          <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/Human_Practices">Human Practices</a></li>
+          </ul>
+        </li>
          <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
-         <li ><a href="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
+         <li ><a title="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
            <ul>
-             <li ><a href="./Data.htm ">Data 1</a></li>
+             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity" title="Data">Cutinase Activity</a></li>
-             <li ><a href="./Data.htm ">Data 2</a></li>
+             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
-            <li ><a href="./Data.htm ">Data 3</a></li>
+title="Data">Ethylene Glycol</a></li>
-         </ul>
+<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>
+          </ul>
          </li>
          <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
            <ul>
-             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols">Protocols</a></li>
+             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a></li>
-             <li ><a href="./Notebook.htm ">Week 1</a></li>
+             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a></li>
-             <li ><a href="./Notebook.htm ">Week 2</a></li>
+             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a></li>
-            <li ><a href="./Notebook.htm ">Week 3</a></li>
            </ul>
          </li>
@@ Line 381: / Line 1,050: @@
          <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
-         <li ><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a></li>
+         <li ><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
+          <ul>
+            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
+            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Module Engineering</a></li>
+            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
+            <li ><a title="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering </a>
+	   	<ul>
+                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Background</a></li>
+	        <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution">Directed Evolution</a></li>
+                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain">Rational Engineering </a></li>
+		</ul>
+</li>
+          </ul>
+        </li>
          <li ><a href="https://2012.igem.org/Team:UC_Davis/Team" title="Team">Team</a></li>
          <li ><a href="https://2012.igem.org/Team:UC_Davis" title="Home">Home</a></li>
       </ul>
     </div>
+<img src="https://static.igem.org/mediawiki/2012/7/71/UCD_protocols_banner.jpg" width="850" height="228">
+<div id="bodyContent">
+<!-- 1st row four boxes starts here -->
+<div id="myleftrightbox">
+<div id="myleftrightbox"  class="fourboxes-1">
+<a href="https://2012.igem.org/Team:UC_Davis/Notebook"><img src="https://static.igem.org/mediawiki/2012/2/24/UCD_notebook_small_banner.jpg"></a>
+</div>
+<div id="myleftrightbox"  class="spacebox">
+<p></p>
+</div>
+<div id="myleftrightbox"  class="fourboxes-2">
+<a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols"><img src="https://static.igem.org/mediawiki/2012/0/03/UCD_protocols_small_banner.jpg"></a>
+</div>
+<div id="myleftrightbox"  class="spacebox">
+<p></p>
+</div>
+<div id="myleftrightbox"  class="fourboxes-3">
+<a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery"><img src="https://static.igem.org/mediawiki/2012/4/43/UCD_Gallery_small_banner-1.jpg"></a>
+</div>
+<div id="myleftrightbox"  class="spacebox">
+<p></p>
+</div>
+<div id="myleftrightbox"  class="fourboxes-4">
+<a href="https://2012.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2012/1/18/UCD_criteria_small_banner.jpg"></a>
+</div>
+</div>
+<!-- 1st row four boxes ends here -->
+<!-- <div id="sponsorbox" style="width:220px; float:right;"> -->
+<div id="sponsorbox">
+<div id="sponsorbox" class="sponsorfloat">
+<!-- twitter starts here -->
+</script>
+<div id="tweets">
+<center>
+<h2>UCDavis iGEM Tweets</h2>
+</center>
+<script charset="utf-8" src="http://widgets.twimg.com/j/2/widget.js"></script>
+<script>
+new TWTR.Widget({
+  version: 2,
+  type: 'profile',
+  rpp: 10,
+  interval: 30000,
+  width: 203,
+  height: 100,
+  theme: {
+    shell: {
+      background: '#004b85',
+      color: '#ffffff'
+    },
+    tweets: {
+      background: '#004b85',
+      color: '#ffffff',
+      links: '#4aed05'
+    }
-<img src="http://img.photobucket.com/albums/v26/bluemelon/protocols_banner-1.jpg" width="850" height="214">
+  },
+  features: {
+    scrollbar: true,
+    loop: false,
+    live: true,
+    behavior: 'all'
+  }
+}).render().setUser('UCDavisiGEM').start();
-  <div id="bodyContent">
+</script>
-             <div id="contentSub"></div>
+</div>
+<!-- twitter ends here -->
+</div>
-<br>
+<div id="sponsorbox" class="sponsorfloat">
-   <div class="floatbox3">
+<center>
-<h1> Protocols </h1>
+<h2>Our Sponsors</h2>
-<article>
+<a href="http://www.novozymes.com/en/Pages/default.aspx" target="_blank"><img src="https://static.igem.org/mediawiki/2011/2/21/UCD_Novozymes-logo.jpg" width="200"></a>
-These are our protocols.
+</center>
-</article>
-   </div>
-<br>
+<center>
-   <div class="floatbox3">
+<a href="http://engineering.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/f/f6/UCD_CoE.png" width="200" height="40"></a>
-<article>
+</center>
-<hp> Registry Part Distribution Rehydration</hp>
-<br>•Add 20 µL sterile H2O to desired well in distribution plate.
-<br>•Incubate at room temperature for ~10 minutes.
-<br>•Transfer resuspension to microcentrifuge tube.
-</article></div>
-<br><div class="floatbox3">
+<center>
-<article>
+<a href="http://biosci.ucdavis.edu/index_js.html" target="_blank"><img src="https://static.igem.org/mediawiki/2011/b/b1/UCD_biosci_sponsor.jpg" width="200" height="90"></a>
-<hp>Transformations</hp>
+</center>
+<center>
+<a href="http://www.genomecenter.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/1/1b/UCD_Genome_center_sponsor.jpg" width="200" height="60"></a>
+</center>
+<center>
+<a href="http://www.cs.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/6/6b/UCD_Computer_sponsor.jpg" width="200"></a>
+</center>
+<center>
+<a href="http://www.bme.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/4/40/UCD_BME_logo_minimal_copy.png" width="200 height="70"></a>
+</center>
+<center>
+<a href="http://www.fishersci.com" target="_blank"><img src="https://static.igem.org/mediawiki/2011/a/a4/UCD_Fisher_Logo.gif" width="200"></a>
+</center>
+<center>
+<a href="http://www.arcadiabio.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/4/46/UCD_Arcadia_sponsor.jpg
+" width="200"></a>
+</center>
+<center>
+<a href="http://provost.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/8/82/UCD_Provost_sponsor.jpg
+" width="200"></a>
+</center>
+<center>
+<a href="http://www.research.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/9/99/UCD_Research_sponsor.jpg" width="200"></a>
+</center>
+<center>
+<a href="http://ucomm.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/b/b4/UCD_Communications_sponsor.jpg" width="200"></a>
+</center>
+<center>
+<a title="" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/25/UCD_Schultz_sponsor.jpg
+" width="200"></a>
+</center>
+</div>
+</div>
+<div id="myleftbox">
+<div id="myleftbox"  class="smallbox">
+<h1>Protocols</h1>
+<!-- accordion starts here -->
+<div id="firstpane" class="menu_list">
+  <p class="menu_head"> Registry Part Distribution Rehydration </p>
+    <div class="menu_body">
+<ul>
+	<li>Add 20 µL sterile H2O to desired well in distribution plate.</li>
+	<li>Incubate at room temperature for ~10 minutes.</li>
+	<li>Transfer resuspension to microcentrifuge tube.</li>
+</ul>
+    </div>
+  <p class="menu_head"> Transformations</p>
+    <div class="menu_body">
 <p>Materials</p>
-•Competent Cells
+<ul>
-<br>•DNA template
+	<li>Competent Cells</li>
-<br>•800 µL LB
+	<li>DNA template</li>
-<br>•LB+Antibiotic Plates
+	<li>800 µL LB</li>
+	<li>LB+Antibiotic Plates</li>
+</ul>
 <p>Procedure</p>
-•Thaw competent cells on ice.
+<ul>
-<br>•Transfer 50 µL of competent cells to chilled falcon tubes.
+	<li>Thaw competent cells on ice.</li>
-<br>•Add 1 µL of template to cells (2.5 µL if dilute).
+	<li>Transfer 50 µL of competent cells to chilled falcon tubes.</li>
-<br>•Incubate on ice for 30 minutes.
+	<li>Add 1 µL of template to cells (2.5 µL if dilute).</li>
-<br>•Heat schock in 42 °C water bath for 90 seconds.
+	<li>Incubate on ice for 30 minutes.</li>
-<br>•Immediately place back onto ice for 2 minutes.
+	<li>Heat schock in 42 °C water bath for 90 seconds.</li>
-<br>•Add 800 µL of LB to each tube.
+	<li>Immediately place back onto ice for 2 minutes.</li>
-<br>•Incubate at 37 °C for 1 hour.
+	<li>Add 800 µL of LB to each tube.</li>
-<br>•Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.
+	<li>Incubate at 37 °C for 1 hour.</li>
-<br>•Incubate overnight at 37 °C.
+	<li>Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.</li>
-</article>
+	<li>Incubate overnight at 37 °C.</li>
-   </div>
+</ul>
+    </div>
-<br><div class="floatbox3">
+  <p class="menu_head"> Restriction Enzyme Double Digest</p>
-<article>
+    <div class="menu_body">
-<hp>Restriction Enzyme Double Digest</hp>
 <p>Materials</p>
-•22 µL dH20
+<ul>
-<br>•1 µL BSA
+	<li>22 µL dH20
-<br>•5 µLBuffer
+	<li>1 µL BSA
-<br>•20 µL Template
+	<li>5 µLBuffer
-<br>•1 µL Enzyme 1
+	<li>20 µL Template
-<br>•1 µL Enzyme 2
+	<li>1 µL Enzyme 1
+	<li>1 µL Enzyme 2
+</ul>
 <p>Procedure</p>
-•Mix reactants, adding enzyme last.
+<ul>
-<br>•Place at 37 C for 3-5 hours.
+	<li>Mix reactants, adding enzyme last.
-<br>•If not purifying or running on a gel immediately, increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
+	<li>Place at 37 °C for 3-5 hours.
-<br>•Run on a gel and extract product.
+	<li>If not purifying or running on a gel immediately, increase to 80 °C for 20 minutes to kill enzymes (some enzymes need only a 65 °C heatkill, check enzyme).
-</article></div>
+	<li>Run on a gel and extract product.
+</ul>
+    </div>
-<br><div class="floatbox3">
+  <p class="menu_head"> PCR (sequence dependent)</p>
-<article>
+    <div class="menu_body">
-<hp>PCR (sequence dependent)</hp>
 <p>Materials</p>
-•10 uL Q Solution
+<ul>
-<br>•5 µL 10x Buffer
+	<li>10 uL Q Solution
-<br>•1.25 µL 10mM dNTPs
+	<li>5 µL 10x Buffer
-<br>•1 µL Template
+	<li>1.25 µL 10mM dNTPs
-<br>•1 µL Forward Primer
+	<li>1 µL Template
-<br>•1 µL Reverse Primer
+	<li>1 µL Forward Primer
-<br>•0.3 µL Taq
+	<li>1 µL Reverse Primer
-<br>•0.15 µL PFU
+	<li>0.3 µL Taq
-<br>•30 µL dH20
+	<li>0.15 µL PFU
+	<li>30 µL dH20
+</ul>
 <p>Procedure</p>
-•Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
+<ul>
-<br>•Run in thermal cycler.
+	<li>Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
-</article></div>
+	<li>Run in thermal cycler.
+</ul>
+   </div>
-<br><div class="floatbox3">
+  <p class="menu_head">PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension)</p>
-<article>
+    <div class="menu_body">
-<hp>PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension) </hp>
 <p>Materials</p>
-•10 µL Q Solution
+<ul>
-<br>•5 µL 10x Buffer
+	<li>10 µL Q Solution
-<br>•1.25 µL 10mM dNTPs
+	<li>5 µL 10x Buffer
-<br>•2.5 µL Forward Primer
+	<li>1.25 µL 10mM dNTPs
-<br>•2.5 µL Reverse Primer
+	<li>2.5 µL Forward Primer
-<br>•0.3 µL Taq
+	<li>2.5 µL Reverse Primer
-<br>•0.15 µL PFU
+	<li>0.3 µL Taq
-<br>• Template based on concentrations determined by SOEing calculator: “Link”
+	<li>0.15 µL PFU
-<br>• ±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”
+	<li>Template based on concentrations determined by SOEing calculator: “Link”
+	<li>±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”
+</ul>
 <p>Procedure</p>
-•Mix reactants into PCR tubes.
+<ul>
-<br>•Run in thermal cycler.
+	<li>Mix reactants into PCR tubes.
-<br>•Continue PCR SOEing of parts until completed.
+	<li>Run in thermal cycler.
-</article></div>
+	<li>Continue PCR SOEing of parts until completed.
-</article></div>
+</ul>
+    </div>
-<br><div class="floatbox3">
+  <p class="menu_head"> Ligation</p>
-<article>
+    <div class="menu_body">
-<hp>Ligation</hp>
 <p>Materials</p>
-•Digested Vector
+<ul>
-<br>•Digested Insert
+	<li>Digested Vector
-<br>•Water
+	<li>Digested Insert
-<br>•T4 DNA Ligase
+	<li>Water
-<br>•T4 DNA Ligase Buffer
+	<li>T4 DNA Ligase
+	<li>T4 DNA Ligase Buffer
+</ul>
 <p>Procedure</p>
-•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>.
+<ul>
-</article></div>
+	<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>.
+</ul>
+    </div>
-<br><div class="floatbox3">
+  <p class="menu_head"> Ethylene Glycol Media</p>
-<article>
+    <div class="menu_body">
-<hp>Ethylene Glycol Media</hp>
 <p>Materials</p>
-•34 mM NaH<sub>2</sub>PO<sub>4</sub>
+<ul>
-<br>•64 mM K<sub>2</sub>HPO<sub>4</sub>
+	<li>34 mM NaH<sub>2</sub>PO<sub>4</sub>
-<br>•20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
+	<li>64 mM K<sub>2</sub>HPO<sub>4</sub>
-<br>•1 µM FeSO<sub>4</sub>
+	<li>20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
-<br>•0.1 mM MgSO<sub>4</sub>
+	<li>1 µM FeSO<sub>4</sub>
-<br>•10 µM CaCl<sub>2</sub>
+	<li>0.1 mM MgSO<sub>4</sub>
-<br>•30 mM Ethylene Glycol
+	<li>10 µM CaCl<sub>2</sub>
-<br>•30 mM Glycolate
+	<li>30 mM Ethylene Glycol
-<br>•0.5% wt/vol Casein Acid Hydrolysate
+	<li>30 mM Glycolate
-<br>•1.5% wt/vol Agar (if making solid media)
+	<li>0.5% wt/vol Casein Acid Hydrolysate
+	<li>1.5% wt/vol Agar (if making solid media)
+</ul>
 <p>Procedure</p>
-•Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.
+<ul>
-<br>•Mix together NaH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, FeSO<sub>4</sub>, MgSO<sub>4</sub>, and CaCl<sub>2</sub>.
+	<li>Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.
-<br>•Titrate to pH 7 with HCl.
+	<li>Mix together NaH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, FeSO<sub>4</sub>, MgSO<sub>4</sub>, and CaCl<sub>2</sub>.
-<br>•Add the glycolate and casein acid hydrolysate.
+	<li>Titrate to pH 7 with HCl.
-<br>•Mix in the ethylene glycol in a fume hood.
+	<li>Add the glycolate and casein acid hydrolysate.
-<br>•If making solid media, also mix in agar.
+	<li>Mix in the ethylene glycol in a fume hood.
-<br>•Autoclave on an appropriate cycle.
+	<li>If making solid media, also mix in agar.
-</article></div>
+	<li>Autoclave on an appropriate cycle.
+</ul>
+    </div>
-<br><div class="floatbox3">
+  <p class="menu_head">Ethylene Glycol Toxicity Assay</p>
-<article>
+    <div class="menu_body">
-<hp>Ethylene Glycol Toxicity Assay</hp>
 <p>Materials</p>
-• Luria Broth Media
+<ul>
-<br>• Ethylene Glycol
+	<li>Luria Broth Media
-<br>• Tecan M200 Pro
+	<li>Ethylene Glycol
-<br>• E. <I>coli</I>
+	<li>Tecan M200 Pro
+	<li>E. <I>coli</I>
+</ul>
+<p>Procedure</p>
+<ul>
+	<li>Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.
+	<li>Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
+	<li>Incubate at 37°C overnight on a shaker at 150 rpm.
+	<li>From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
+	<li>Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
+	<li>Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.
+	<li>Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
+	<li>With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.
+	<li>Add “blank” wells of just LB media to an entire column to serve as a control for the LB.
+	<li>Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.
+	<li>Aliquot the remaining dilutions as the diagram below depicts.
+	<li>Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.
+	<li>Follow the steps of your Tecan machine appropriate to the specific bacterial strain.
+</ul>
+    </div>
+  <p class="menu_head"> EMS (Ethyl methanesulfonate Mutagenesis)</p>
+    <div class="menu_body">
+<p>Materials</p>
+<ul>
+<li>Buffer A: Minimal medium + 0.2 M Tris (pH 7.5). per liter:
+	<ul>
+	<li>K<sub>2</sub>HPO<sub>4</sub>: 10.5g</li>
+	<li>KH<sub>2</sub>PO<sub>4</sub>: 4.5g</li>
+	<li>(NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>: 1g</li>
+	<li>Sodium Citrate * 2H<sub>2</sub>O: 0.5g</li>
+	<li>1M Tris (pH 7.5): 200mL Tris</li>
+	</ul>
+</li>
+</ul>
+<sub>Obtained components from “Miller,  J. H. (1972) Experiments  in Molecular Genetics 	(Cold Spring Harbor Lab., Cold Spring Harbor, NY)” pg 432</sub>
+<ul>
+<li>EMS (Sigma)</li>
+<li>50mL conical Corning tubes</li>
+<li>15mL Falcon tubes</li>
+<li>LB Broth</li>
+<li>Ethylene Glycol Agar Plates</li>
+</ul>
 <p>Procedure</p>
-• Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.
+<ul>
-<br>1.	Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
+	<li>1.	Prepare a 10mL liquid culture in LB in a 50mL conical tube and grow it overnight until it reaches OD 0.2.</li>
-<br>2.	Incubate at 37°C overnight on a shaker at 150 rpm.
+	<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
-<br>3.	From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
+	<li>Wash twice with 10mL Buffer A.</li>
-<br>4.	Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
+            <ul>
-<br>5.	Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.
+                <li>Pipet up and down to mix, pellet cells, decant supernatant.</li>
-<br>6.	Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
+            </ul>
-<br>7.	With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.
+	<li>Re-suspend the pellet in 5mL of Buffer A and transfer the medium to a Falcon tube.</li>
-<br>8.	Add “blank” wells of just LB media to an entire column to serve as a control for the LB.
+	<li>Add (35μL, 70μL, or 105μL) of EMS into each tube of re-suspended cells. </li>
-<br>9.	Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.
+	<li>Close tubes and parafilm the lid 2X.</li>
-<br>10.	Aliquot the remaining dilutions as the diagram below depicts.
+	<li>Vortex to mix and place in a secondary container (50mL conical tube).</li>
-<br>11.	Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.
+	<li>Transfer to mixing platform and agitate at ~30 rpm at 37°C.</li>
-<br>12.	Follow the steps of your Tecan machine appropriate to the specific bacterial strain.
+	<li>Withdraw samples at fixed time points.</li>
+	<li>At each time point, take 1mL aliquots of the sample and place in a 15mL Falcon tube. Place in a secondary container (50mL Conical tube) and spin at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 5 minutes. </li>
+	<li>Discard supernatant in waste container. </li>
+	<li>Wash twice in 5mL of Buffer A and discard supernatant in waste container.</li>
+	<li>Re-suspend in 5mL of Buffer A and titer for viable cells.</li>
+	<li>Add 0.5mL of mutagenized culture into 10mL of LB broth in a 50mL conical tube.</li>
+	<li>Grow cultures overnight at 37°C and plate for mutants on EG agar plates.</li>
+	<li>Look for viable cells</li>
+</ul>
-</article></div>
+<p>Safety: Health Hazards</p>
+<ul>
+	<li>Acute toxicity (oral, dermal, inhalation), category 4 </li>
+	<li>Skin irritation, category 2 </li>
+	<li>Eye irritation, category 2 </li>
+	<li>Skin sensitization, category 1 </li>
+	<li>Specific Target Organ Toxicity – Single exposure, category 3</li>
+	<li>LD50 – 470mg/kg</li>
+</ul>
+<p>Recommended Safety & Handling</p>
+<ul><li>Always work in fume hood and wear lab coat, goggles, and gloves.</li></ul>
+    </div>
+<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>
-<script>if (window.runOnloadHook) runOnloadHook();</script>
+<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>
+<!-- accordion ends here -->
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+<div id="myleftbox"  class="smallboxsite">
+<ul style="font-size:10px;list-style-image:none;list-style-type:none;float:left;display:inline;color:#000000;"
+ >
+<li style="float:left;margin:0 10px;"><a
+href="https://2012.igem.org/Team:UC_Davis"><p>Home</p><ul
+style="text-indent:-15px;
+list-style-image:none;list-style-type:none;color:#000000;"><li><a
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+href="https://2012.igem.org/Team:UC_Davis/Team"><p>Team</p><ul
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+href="https://2012.igem.org/Team:UC_Davis/Project "><p>Project</p></a>
+<ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000"><li><a
+style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a>
+</li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst ">Module
+Engineering</a></li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering
+">Protein Engineering</a></li>
+<li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Project/Strain ">Chassis
+Engineering</a></li>
+   <li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution ">
+- Directed Evolution</a></li>
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+href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain "> -
+Rational Engineering</a></li>
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+href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a>
+</li></ul> </li>
+<li style="float:left ;margin:0 10px"><a
+href="https://2012.igem.org/Team:UC_Davis/Safety "> <p>Safety</p></a>
+<a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Safety "> Safety</a> </li>
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+href="https://2012.igem.org/Team:UC_Davis/Notebook ">
+<p>Notebook</p></a> <ul
+style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
+ "><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a>
+</li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols
+">Protocols</a> </li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a>
+</li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a> </li>
+</ul> </li>
+<li style="float:left ;margin:0 10px;"><a
+title="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p></a> <ul
+style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
+ "><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity ">
+Cutinase Activity</a> </li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol ">
+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>
+<li style="float:left ;margin:0 10px"><a
+href="https://2012.igem.org/Team:UC_Davis/Attributions ">
+<p>Attribution </p></a><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Attributions ">
+Attribution</a></li>
+<li style="float:left ;margin:0 10px"><a
+href="https://2012.igem.org/Main_Page "> <p>iGEM </p></a><ul
+style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
+ "><li><a style="color:#000000 " href="https://2012.igem.org/Main_Page
+">Main iGEM</a> </li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Criteria "> Criteria</a>
+</li><li><a style="color:#000000 "
+href="https://2012.igem.org/Team:UC_Davis/Human_Practices ">Human
+Practices</a></li> </ul>
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Team:UC Davis/Notebook/Protocols

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Latest revision as of 02:52, 27 October 2012

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