Difference between revisions of "Team:NCKU Tainan/Protocol"

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             <a class="btn col-md-12" data-toggle="collapse" href="#test" role="button" aria-expanded="false"
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             <a class="btn col-md-12" data-toggle="collapse" href="#Plasmid_Construction" role="button" aria-expanded="false"
 
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                 aria-controls="multiCollapseExample1">
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                 test
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                 Plasmid Construction
 
                 <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
 
                 <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
 
             </a>
 
             </a>
 
         </div>
 
         </div>
  
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         <div class="collapse multi-collapse" id="test"">
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         <div class="collapse multi-collapse" id="Plasmid_Construction">
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        <p class="pcontent">1. Digestion (vector)</p>
 +
        <table class="card card-body">
 +
    　<tr>
 +
        　<th>Plasmid</th>
 +
        　<th>200 ng</th>
 +
        <th>1000 ng</th>
 +
    </tr>
  
 +
    <tr>
 +
        　<td>EcoRI / SpeI</td>
 +
        　<td>0.2 μl</td>
 +
        <td>1 μl</td>
 +
    </tr>
 +
 +
    　<tr>
 +
        　<td>XbaI / PstI</td>
 +
        　<td>0.2 μl</td>
 +
        <td>1 μl</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>CutSmart Buffer</td>
 +
        　<td>2 μl</td>
 +
        <td>5 μl</td>
 +
    </tr>
 +
 +
    　<tr>
 +
        　<td>ddH2O</td>
 +
        　<td>Up to 20 μl</td>
 +
        <td>Up to 50 μl</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>Digestion at 37℃ for 2.5hr.</td>
 +
        　<td></td>
 +
        <td></td>
 +
    </tr>
 +
   
 +
</table>
 +
 +
          <p class="pcontent">2. Digestion (insert)</p>
 +
          <table class="card card-body">
 +
    　<tr>
 +
        　<th>Plasmid</th>
 +
        　<th>200 ng</th>
 +
        <th>1000 ng</th>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>EcoRI / XbaI</td>
 +
        　<td>0.2 μl</td>
 +
        <td>1 μl</td>
 +
    </tr>
 +
 +
    　<tr>
 +
        　<td>SpeI  / PstI</td>
 +
        　<td>0.2 μl</td>
 +
        <td>1 μl</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>CutSmart Buffer</td>
 +
        　<td>2 μl</td>
 +
        <td>5 μl</td>
 +
    </tr>
 +
 +
    　<tr>
 +
        　<td>ddH2O</td>
 +
        　<td>Up to 20 μl</td>
 +
        <td>Up to 50 μl</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>Digestion at 37℃ for 2.5hr.</td>
 +
        　<td></td>
 +
        <td></td>
 +
    </tr>
 +
       
 +
</table>
 +
 +
        <p class="pcontent">3.Confirm the size of the digested product by gel electrophoresis.
 +
        4. Gel purification of the target size.
 +
        5. Ligation
 +
 +
        <table class="card card-body">
 +
 +
    　<tr>
 +
        　<td>Vector (2 kbp)</td>
 +
        　<td rowspan="2">molar ratio = 1:3</br>(can up to 1:10 depending on the DNA sizes)</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>Insert (1.5 kbp)</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>Quick Ligase Reaction Buffer (2X)*</td>
 +
          <td>10 μl</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>Quick Ligase</td>
 +
          <td>1 μl</td>
 +
    </tr>
 +
 +
    <tr>
 +
        　<td>ddH2O</td>
 +
        <td>Up to 20 μl</td>
 +
    </tr>
 +
 +
</table>
 +
 +
       
  
 
         </div>
 
         </div>

Revision as of 15:30, 21 September 2018

Protocol




1. Gently mix the following reaction by pipetting and centrifuge briefly.

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　
20 μl system 50 μl system
Template12～20 ng 30～50 ng
Forward primer1.0 μl 2.5 μl
Reverse primer1.0 μl 2.5 μl
dNTP1.6 μl 4.0 μl
10x Buffer2.0 μl 5.0 μl


Program of KOD DNA polymerase

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　 　 　
TemperatureTime
94 ℃3 min. 25～30 cycles
94 ℃
(Denaturation)		40 sec
57.5 ℃
(Annealing)		30 sec
72 ℃
(Extension)		Depend on sequence size
(2 kbp/min. for Taq)
72 ℃5 min.
4 ℃∞


2. Confirm the size of the digested product by gel electrophoresis.

3. Gel purification of the target size.


1. Digestion (vector)

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　
Plasmid200 ng 1000 ng
EcoRI / SpeI0.2 μl 1 μl
XbaI / PstI0.2 μl 1 μl
CutSmart Buffer2 μl 5 μl
ddH2OUp to 20 μl Up to 50 μl
Digestion at 37℃ for 2.5hr.

2. Digestion (insert)

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　
Plasmid200 ng 1000 ng
EcoRI / XbaI0.2 μl 1 μl
SpeI / PstI0.2 μl 1 μl
CutSmart Buffer2 μl 5 μl
ddH2OUp to 20 μl Up to 50 μl
Digestion at 37℃ for 2.5hr.

3.Confirm the size of the digested product by gel electrophoresis. 4. Gel purification of the target size. 5. Ligation

　 　 　 　 　 　 　
Vector (2 kbp)molar ratio = 1:3
(can up to 1:10 depending on the DNA sizes)
Insert (1.5 kbp)
Quick Ligase Reaction Buffer (2X)* 10 μl
Quick Ligase 1 μl
ddH2O Up to 20 μl

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