Difference between revisions of "GDSYZX-United/NOTEBOOK/protocol"

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}
 
}
 
nav {padding-top:10px;}
 
nav {padding-top:10px;}
 +
.text_font {
 +
line-height:120%;
 +
font-size:130%
 +
}
 
</style>
 
</style>
 
</head>
 
</head>
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<a aria-expanded="false" role="button" href="#" class="dropdown-toggle" data-toggle="dropdown"> TEAM <span class="caret"></span></a>
 
<a aria-expanded="false" role="button" href="#" class="dropdown-toggle" data-toggle="dropdown"> TEAM <span class="caret"></span></a>
 
<ul role="menu" class="dropdown-menu">
 
<ul role="menu" class="dropdown-menu">
−
<li><a href="#">Team members</a></li>
+
<li><a href="https://2016.igem.org/GDSYZX-United/TEAM/team_members">Team members</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
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</li>
 
</li>
 
<li class="dropdown">
 
<li class="dropdown">
−
<a aria-expanded="false" role="button" href="#"> SAFETY </a>
+
<a aria-expanded="false" role="button" href="#" class="dropdown-toggle" data-toggle="dropdown"> SAFETY <span class="caret"></span></a>
 +
<ul role="menu" class="dropdown-menu">
 +
<li><a href="https://2016.igem.org/GDSYZX-United/SAFETY/lab_safety">Lab safety</a></li>
 +
<li><a href="https://2016.igem.org/GDSYZX-United/SAFETY/safety_of_process">Safety_of_process</a></li>
 +
</ul>
 
</li>
 
</li>
 
<li class="dropdown">
 
<li class="dropdown">
Line 95: Line 103:
 
                 <div class="ibox float-e-margins">
 
                 <div class="ibox float-e-margins">
 
                     <div class="timeline">
 
                     <div class="timeline">
 +
<div class="timeline-item">
 +
                            <div class="row">
 +
                                <div class="col-xs-12 text-center">
 +
                                    <h1>Protocol</h1>
 +
                                </div>
 +
                            </div>
 +
                        </div>
 
                         <div class="timeline-item">
 
                         <div class="timeline-item">
 
                             <div class="row">
 
                             <div class="row">
−
                                 <div class="col-xs-3 date">
+
                                 <div class="col-xs-3 date"></div>
−
</div>
+
 
                                 <div class="col-xs-8 content">
 
                                 <div class="col-xs-8 content">
−
                                     <p class="m-b-xs"><strong>1.Extract Arabidopsis genomic DNA</strong>
+
                                     <div class="m-b-xs text_font"><strong>1.Extract Arabidopsis genomic DNA</strong></div>
−
                                    </p>
+
                                     <div class="text_font">Material:</div>
−
                                     <p>Material:<br>
+
<ul>
−
&emsp;&emsp;EZ-10 Spin Column Plant Genomic DNA Purification Kit<br>
+
<li><div class="text_font">EZ-10 Spin Column Plant Genomic DNA Purification Kit</div></li>
−
&emsp;&emsp;β-mercaptoethanol<br>
+
<li><div class="text_font">β-mercaptoethanol</div></li>
−
&emsp;&emsp;RNaseA<br>
+
<li><div class="text_font">RNaseA</div></li>
−
&emsp;&emsp;Chloroform<br>
+
<li><div class="text_font">Chloroform</div></li>
−
&emsp;&emsp;Ice box and ice<br>
+
<li><div class="text_font">Ice box and ice</div></li>
−
</p>
+
</ul>
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
Line 117: Line 131:
 
</div>
 
</div>
 
                                 <div class="col-xs-8 content">
 
                                 <div class="col-xs-8 content">
−
                                     <p class="m-b-xs"><strong>2.PCR</strong>
+
                                     <div class="m-b-xs text_font"><strong>2.PCR</strong></div>
−
                                    </p>
+
                                     <div class="text_font">Material:</div>
−
                                     <p>Material:<br>
+
<ul>
−
&emsp;&emsp;ddH20<br>
+
<li><div class="text_font">ddH20</div></li>
−
&emsp;&emsp;dNTPs mix<br>
+
<li><div class="text_font">dNTPs mix</div></li>
−
&emsp;&emsp;10×Buffer<br>
+
<li><div class="text_font">10×Buffer</div></li>
−
&emsp;&emsp;Taq polymerase<br>
+
<li><div class="text_font">Taq polymerase</div></li>
−
&emsp;&emsp;PCR Primers<br>
+
<li><div class="text_font">PCR Primers</div></li>
−
&emsp;&emsp;Arabidopsis genomic DNA<br>
+
<li><div class="text_font">Arabidopsis genomic DNA</div></li>
−
Methods:<br>
+
</ul>
−
&emsp;&emsp;1.add material into a 0.2ml EP tube according to the table<br>
+
<div class="text_font">Methods:</div>
−
<table>
+
<ol>
−
<thead>
+
<li>
−
<tr>
+
<div class="text_font">add material into a 0.2ml EP tube according to the table</div>
−
<th>Material</th>
+
<table class="table">
−
<th>dosage/μl</th>
+
<thead>
−
</tr>
+
<tr>
−
</thead>
+
<th>Material</th>
−
<tbody>
+
<th>dosage/μl</th>
−
<tr>
+
</tr>
−
<td>dNTPs mix</td>
+
</thead>
−
<td>1</td>
+
<tbody>
−
</tr>
+
<tr>
−
<tr>
+
<td>dNTPs mix</td>
−
<td>Forward primer</td>
+
<td>1</td>
−
<td>0.5</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>Forward primer</td>
−
<td>Reverse primer</td>
+
<td>0.5</td>
−
<td>0.5</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>Reverse primer</td>
−
<td>Arabidopsis genomic DNA</td>
+
<td>0.5</td>
−
<td>3</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>Arabidopsis genomic DNA</td>
−
<td>ddH20</td>
+
<td>3</td>
−
<td>12</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>ddH20</td>
−
<td>Taq  polymerase</td>
+
<td>12</td>
−
<td>0.5</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>Taq  polymerase</td>
−
<td>Total</td>
+
<td>0.5</td>
−
<td>20</td>
+
</tr>
−
</tr>
+
<tr>
−
</tbody>
+
<td>Total</td>
−
</table>
+
<td>20</td>
−
<p>&emsp;&emsp;2.set the PCR program <br>
+
</tr>
−
For promoter cop1,phyB,pif1,gene hhl1,gene flu:<br>
+
</tbody>
−
94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)<br>
+
</table>  
−
For pHhl1-gene hhl1:<br>
+
</li>
−
94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)<br>
+
<li>
−
Tm of each primer:<br></p>
+
<div class="text_font">set the PCR program </div>
−
<table>
+
<ul>
−
<thead>
+
<li>
−
<tr>
+
<div class="text_font">For promoter cop1,phyB,pif1,gene hhl1,gene flu:</div>
−
<th>Name</th>
+
<div class="text_font">94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)</div>
−
<th>Tm(Forward/Reverse)(℃)</th>
+
</li>
−
<th>Tm in PCR(℃)</th>
+
<li>
−
</tr>
+
<div class="text_font">For pHhl1-gene hhl1:</div>
−
</thead>
+
<div class="text_font">94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)</div>
−
<tbody>
+
</li>
−
<tr>
+
<li>
−
<td>Promoter pif1</td>
+
<div class="text_font">Tm of each primer: </div>
−
<td>62.59/62.67</td>
+
<table class="table">
−
<td>63</td>
+
<thead>
−
</tr>
+
<tr>
−
<tr>
+
<th>Name</th>
−
<td>Promoter cop1</td>
+
<th>Tm(Forward/Reverse)(℃)</th>
−
<td>66.77/65.46</td>
+
<th>Tm in PCR(℃)</th>
−
<td>66</td>
+
</tr>
−
</tr>
+
</thead>
−
<tr>
+
<tbody>
−
<td>Promoter phyB</td>
+
<tr>
−
<td>63.94/63.90</td>
+
<td>Promoter pif1</td>
−
<td>64</td>
+
<td>62.59/62.67</td>
−
</tr>
+
<td>63</td>
−
<tr>
+
</tr>
−
<td>pHhl1-genehhl1</td>
+
<tr>
−
<td>60.75/59.38</td>
+
<td>Promoter cop1</td>
−
<td>60</td>
+
<td>66.77/65.46</td>
−
</tr>
+
<td>66</td>
−
<tr>
+
</tr>
−
<td>gene hhl1</td>
+
<tr>
−
<td>60.90/59.38</td>
+
<td>Promoter phyB</td>
−
<td>60</td>
+
<td>63.94/63.90</td>
−
</tr>
+
<td>64</td>
−
<tr>
+
</tr>
−
<td>Fluorescent gene flu</td>
+
<tr>
−
<td>62.42/66.90</td>
+
<td>pHhl1-genehhl1</td>
−
<td>65</td>
+
<td>60.75/59.38</td>
−
</tr>
+
<td>60</td>
−
</tbody>
+
</tr>
−
</table>
+
<tr>
−
</p>
+
<td>gene hhl1</td>
 +
<td>60.90/59.38</td>
 +
<td>60</td>
 +
</tr>
 +
<tr>
 +
<td>Fluorescent gene flu</td>
 +
<td>62.42/66.90</td>
 +
<td>65</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</li>
 +
</ul>
 +
</li>
 +
</ol>
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
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</div>
 
</div>
 
                                 <div class="col-xs-8 content">
 
                                 <div class="col-xs-8 content">
−
                                     <p class="m-b-xs"><strong>3.digestion</strong>
+
                                     <div class="m-b-xs text_font"><strong>3.digestion</strong></div>
−
                                    </p>
+
                                     <div class="text_font">Material:</div>
−
                                     <p>Material:<br>
+
<ul>
−
&emsp;&emsp;ddH20<br>
+
<li><div class="text_font">ddH20</div></li>
−
&emsp;&emsp;BsaⅠBuffer<br>
+
<li><div class="text_font">BsaⅠBuffer</div></li>
−
&emsp;&emsp;PstⅠBuffer<br>
+
<li><div class="text_font">PstⅠBuffer</div></li>
−
&emsp;&emsp;restriction enzyme BsaⅠ，PstⅠ，EcorⅠ<br>
+
<li><div class="text_font">restriction enzyme BsaⅠ，PstⅠ，EcorⅠ</div></li>
−
&emsp;&emsp;PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9<br>
+
<li><div class="text_font">PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9</div></li>
−
&emsp;&emsp;Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9<br>
+
<li><div class="text_font">Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9</div></li>
−
Method：<br>
+
</ul>
−
&emsp;&emsp;1.add materials into a 1.5ml EP tube according to the table below:</p>
+
<div class="text_font">Method</div>
−
<table>
+
<ol>
−
<thead>
+
<li>
−
<tr>
+
<div class="text_font">1.add materials into a 1.5ml EP tube according to the table below:</div>
−
<th>Material</th>
+
<table class="table">
−
<th>dosage/μl</th>
+
<thead>
−
</tr>
+
<tr>
−
</thead>
+
<th>Material</th>
−
<tbody>
+
<th>dosage/μl</th>
−
<tr>
+
</tr>
−
<td>PCR  product(after purification)</td>
+
</thead>
−
<td>17</td>
+
<tbody>
−
</tr>
+
<tr>
−
<tr>
+
<td>PCR  product(after purification)</td>
−
<td>restriction enzyme buffer</td>
+
<td>17</td>
−
<td>3 (/4)</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>restriction enzyme buffer</td>
−
<td>restriction enzyme</td>
+
<td>3 (/4)</td>
−
<td>1(0.5 each)</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>restriction enzyme</td>
−
<td>ddH20</td>
+
<td>1(0.5 each)</td>
−
<td>4</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>ddH20</td>
−
<td>Total</td>
+
<td>4</td>
−
<td>25</td>
+
</tr>
−
</tr>
+
<tr>
−
</tbody>
+
<td>Total</td>
−
</table>
+
<td>25</td>
−
<table>
+
</tr>
−
<thead>
+
</tbody>
−
<tr>
+
</table>
−
<th>PCR product</th>
+
<br>
−
<th>cutting sites</th>
+
<table class="table">
−
<th>restriction enzyme Buffer</th>
+
<thead>
−
</tr>
+
<tr>
−
</thead>
+
<th>PCR product</th>
−
<tbody>
+
<th>cutting sites</th>
−
<tr>
+
<th>restriction enzyme Buffer</th>
−
<td>pHhl1-hhl1</td>
+
</tr>
−
<td>BsaⅠ+EcorⅠ</td>
+
</thead>
−
<td>3μlPstⅠ+1μlBsaⅠ</td>
+
<tbody>
−
</tr>
+
<tr>
−
<tr>
+
<td>pHhl1-hhl1</td>
−
<td>hhl1</td>
+
<td>BsaⅠ+EcorⅠ</td>
−
<td>PstⅠ+EcorⅠ</td>
+
<td>3μlPstⅠ+1μlBsaⅠ</td>
−
<td>3μlPstⅠ</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>hhl1</td>
−
<td>flu</td>
+
<td>PstⅠ+EcorⅠ</td>
−
<td>EcorⅠ+BsaⅠ</td>
+
<td>3μlPstⅠ</td>
−
<td>3μlPstⅠ+1μlBsa1Ⅰ</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>flu</td>
−
<td>cop1</td>
+
<td>EcorⅠ+BsaⅠ</td>
−
<td>BsaⅠ+PstⅠ</td>
+
<td>3μlPstⅠ+1μlBsa1Ⅰ</td>
−
<td>3μlPstⅠ</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>cop1</td>
−
<td>phyB</td>
+
<td>BsaⅠ+PstⅠ</td>
−
<td>BsaⅠ+PstⅠ</td>
+
<td>3μlPstⅠ</td>
−
<td>3μlPstⅠ</td>
+
</tr>
−
</tr>
+
<tr>
−
<tr>
+
<td>phyB</td>
−
<td>pif1</td>
+
<td>BsaⅠ+PstⅠ</td>
−
<td>BsaⅠ+PstⅠ</td>
+
<td>3μlPstⅠ</td>
−
<td>3μlPstⅠ</td>
+
</tr>
−
</tr>
+
<tr>
−
</tbody>
+
<td>pif1</td>
−
</table>
+
<td>BsaⅠ+PstⅠ</td>
−
<p>&emsp;&emsp;2.Place the tube in a 37℃ incubator, stand for 1 hour.<br>
+
<td>3μlPstⅠ</td>
−
&emsp;&emsp;3.Water bath the tube in 65℃ for 20 mins to end the digestion reaction.<br>
+
</tr>
−
</p>
+
</tbody>
 +
</table>
 +
</li>
 +
<li><div class="text_font">2.Place the tube in a 37℃ incubator, stand for 1 hour.</div></li>
 +
<li><div class="text_font">3.Water bath the tube in 65℃ for 20 mins to end the digestion reaction.</div></li>
 +
<ol>
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>

Revision as of 15:19, 29 September 2016

Protocol

1.Extract Arabidopsis genomic DNA
Material:
  • EZ-10 Spin Column Plant Genomic DNA Purification Kit
  • β-mercaptoethanol
  • RNaseA
  • Chloroform
  • Ice box and ice
2.PCR
Material:
  • ddH20
  • dNTPs mix
  • 10×Buffer
  • Taq polymerase
  • PCR Primers
  • Arabidopsis genomic DNA
Methods:
  1. add material into a 0.2ml EP tube according to the table
    Material dosage/μl
    dNTPs mix 1
    Forward primer 0.5
    Reverse primer 0.5
    Arabidopsis genomic DNA 3
    ddH20 12
    Taq polymerase 0.5
    Total 20
  2. set the PCR program
    • For promoter cop1,phyB,pif1,gene hhl1,gene flu:
      94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)
    • For pHhl1-gene hhl1:
      94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)
    • Tm of each primer:
      Name Tm(Forward/Reverse)(℃) Tm in PCR(℃)
      Promoter pif1 62.59/62.67 63
      Promoter cop1 66.77/65.46 66
      Promoter phyB 63.94/63.90 64
      pHhl1-genehhl1 60.75/59.38 60
      gene hhl1 60.90/59.38 60
      Fluorescent gene flu 62.42/66.90 65
3.digestion
Material:
  • ddH20
  • BsaⅠBuffer
  • PstⅠBuffer
  • restriction enzyme BsaⅠ，PstⅠ，EcorⅠ
  • PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9
  • Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9
Method
  1. 1.add materials into a 1.5ml EP tube according to the table below:
    Material dosage/μl
    PCR product(after purification) 17
    restriction enzyme buffer 3 (/4)
    restriction enzyme 1(0.5 each)
    ddH20 4
    Total 25

    PCR product cutting sites restriction enzyme Buffer
    pHhl1-hhl1 BsaⅠ+EcorⅠ 3μlPstⅠ+1μlBsaⅠ
    hhl1 PstⅠ+EcorⅠ 3μlPstⅠ
    flu EcorⅠ+BsaⅠ 3μlPstⅠ+1μlBsa1Ⅰ
    cop1 BsaⅠ+PstⅠ 3μlPstⅠ
    phyB BsaⅠ+PstⅠ 3μlPstⅠ
    pif1 BsaⅠ+PstⅠ 3μlPstⅠ
  2. 2.Place the tube in a 37℃ incubator, stand for 1 hour.
  3. 3.Water bath the tube in 65℃ for 20 mins to end the digestion reaction.