Thomasboboto (Talk | contribs) |
Thomasboboto (Talk | contribs) |
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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=" | + | <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"> | ||
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<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 class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
− | < | + | <div class="m-b-xs text_font"><strong>1.Extract Arabidopsis genomic DNA</strong></div> |
− | + | <div class="text_font">Material:</div> | |
− | < | + | <ul> |
− | + | <li><div class="text_font">EZ-10 Spin Column Plant Genomic DNA Purification Kit</div></li> | |
− | + | <li><div class="text_font">β-mercaptoethanol</div></li> | |
− | + | <li><div class="text_font">RNaseA</div></li> | |
− | + | <li><div class="text_font">Chloroform</div></li> | |
− | + | <li><div class="text_font">Ice box and ice</div></li> | |
− | + | </ul> | |
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
− | < | + | <div class="m-b-xs text_font"><strong>2.PCR</strong></div> |
− | + | <div class="text_font">Material:</div> | |
− | < | + | <ul> |
− | + | <li><div class="text_font">ddH20</div></li> | |
− | + | <li><div class="text_font">dNTPs mix</div></li> | |
− | + | <li><div class="text_font">10×Buffer</div></li> | |
− | + | <li><div class="text_font">Taq polymerase</div></li> | |
− | + | <li><div class="text_font">PCR Primers</div></li> | |
− | + | <li><div class="text_font">Arabidopsis genomic DNA</div></li> | |
− | Methods:< | + | </ul> |
− | + | <div class="text_font">Methods:</div> | |
− | + | <ol> | |
− | + | <li> | |
− | + | <div class="text_font">add material into a 0.2ml EP tube according to the table</div> | |
− | + | <table class="table"> | |
− | + | <thead> | |
− | + | <tr> | |
− | + | <th>Material</th> | |
− | + | <th>dosage/μl</th> | |
− | + | </tr> | |
− | + | </thead> | |
− | + | <tbody> | |
− | + | <tr> | |
− | + | <td>dNTPs mix</td> | |
− | + | <td>1</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Forward primer</td> | |
− | + | <td>0.5</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Reverse primer</td> | |
− | + | <td>0.5</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Arabidopsis genomic DNA</td> | |
− | + | <td>3</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH20</td> | |
− | + | <td>12</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Taq polymerase</td> | |
− | + | <td>0.5</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Total</td> | |
− | + | <td>20</td> | |
− | + | </tr> | |
− | + | </tbody> | |
− | + | </table> | |
− | + | </li> | |
− | + | <li> | |
− | + | <div class="text_font">set the PCR program </div> | |
− | + | <ul> | |
− | + | <li> | |
− | + | <div class="text_font">For promoter cop1,phyB,pif1,gene hhl1,gene flu:</div> | |
− | + | <div class="text_font">94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)</div> | |
− | + | </li> | |
− | + | <li> | |
− | + | <div class="text_font">For pHhl1-gene hhl1:</div> | |
− | + | <div class="text_font">94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)</div> | |
− | + | </li> | |
− | + | <li> | |
− | + | <div class="text_font">Tm of each primer: </div> | |
− | + | <table class="table"> | |
− | + | <thead> | |
− | + | <tr> | |
− | + | <th>Name</th> | |
− | + | <th>Tm(Forward/Reverse)(℃)</th> | |
− | + | <th>Tm in PCR(℃)</th> | |
− | + | </tr> | |
− | + | </thead> | |
− | + | <tbody> | |
− | + | <tr> | |
− | + | <td>Promoter pif1</td> | |
− | + | <td>62.59/62.67</td> | |
− | + | <td>63</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Promoter cop1</td> | |
− | + | <td>66.77/65.46</td> | |
− | + | <td>66</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Promoter phyB</td> | |
− | + | <td>63.94/63.90</td> | |
− | + | <td>64</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>pHhl1-genehhl1</td> | |
− | + | <td>60.75/59.38</td> | |
− | + | <td>60</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | </ | + | <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"> | ||
− | < | + | <div class="m-b-xs text_font"><strong>3.digestion</strong></div> |
− | + | <div class="text_font">Material:</div> | |
− | < | + | <ul> |
− | + | <li><div class="text_font">ddH20</div></li> | |
− | + | <li><div class="text_font">BsaⅠBuffer</div></li> | |
− | + | <li><div class="text_font">PstⅠBuffer</div></li> | |
− | + | <li><div class="text_font">restriction enzyme BsaⅠ,PstⅠ,EcorⅠ</div></li> | |
− | + | <li><div class="text_font">PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9</div></li> | |
− | + | <li><div class="text_font">Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9</div></li> | |
− | + | </ul> | |
− | + | <div class="text_font">Method</div> | |
− | + | <ol> | |
− | + | <li> | |
− | + | <div class="text_font">1.add materials into a 1.5ml EP tube according to the table below:</div> | |
− | + | <table class="table"> | |
− | + | <thead> | |
− | + | <tr> | |
− | + | <th>Material</th> | |
− | + | <th>dosage/μl</th> | |
− | + | </tr> | |
− | + | </thead> | |
− | + | <tbody> | |
− | + | <tr> | |
− | + | <td>PCR product(after purification)</td> | |
− | + | <td>17</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>restriction enzyme buffer</td> | |
− | + | <td>3 (/4)</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>restriction enzyme</td> | |
− | + | <td>1(0.5 each)</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH20</td> | |
− | + | <td>4</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Total</td> | |
− | + | <td>25</td> | |
− | + | </tr> | |
− | + | </tbody> | |
− | + | </table> | |
− | + | <br> | |
− | + | <table class="table"> | |
− | + | <thead> | |
− | + | <tr> | |
− | + | <th>PCR product</th> | |
− | + | <th>cutting sites</th> | |
− | + | <th>restriction enzyme Buffer</th> | |
− | + | </tr> | |
− | + | </thead> | |
− | + | <tbody> | |
− | + | <tr> | |
− | + | <td>pHhl1-hhl1</td> | |
− | + | <td>BsaⅠ+EcorⅠ</td> | |
− | + | <td>3μlPstⅠ+1μlBsaⅠ</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>hhl1</td> | |
− | + | <td>PstⅠ+EcorⅠ</td> | |
− | + | <td>3μlPstⅠ</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>flu</td> | |
− | + | <td>EcorⅠ+BsaⅠ</td> | |
− | + | <td>3μlPstⅠ+1μlBsa1Ⅰ</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>cop1</td> | |
− | + | <td>BsaⅠ+PstⅠ</td> | |
− | + | <td>3μlPstⅠ</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>phyB</td> | |
− | + | <td>BsaⅠ+PstⅠ</td> | |
− | + | <td>3μlPstⅠ</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>pif1</td> | |
− | + | <td>BsaⅠ+PstⅠ</td> | |
− | + | <td>3μlPstⅠ</td> | |
− | + | </tr> | |
− | < | + | </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:
-
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 -
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.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.Place the tube in a 37℃ incubator, stand for 1 hour.
- 3.Water bath the tube in 65℃ for 20 mins to end the digestion reaction.