Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W10"

 
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Line 372: Line 372:
 
</div>
 
</div>
  
−
<div class="column full_size">
+
<div class="column half_size">
 
<div align="center">
 
<div align="center">
  
Line 383: Line 383:
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> 10X T4 Buffer </td>
+
<td> Ligase Buffer </td>
−
<td> </td>
+
<td> 2 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
<td> Vector </td>
+
<td> Toggle pieces </td>
−
<td> # (# ng) </td>
+
<td> 10 (500 ng/ul) </td>
 
</tr>
 
</tr>
−
<td> Construct </td>
+
<td> Plasmid </td>
−
<td> # (# ng) </td>
+
<td> 2  </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> H<sub>2</sub>O</td>
 
<td> H<sub>2</sub>O</td>
−
<td> # </td>
+
<td> 5 </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td> Ligase </td>
 
<td> Ligase </td>
−
<td> #</td>
+
<td> 1 </td>
 
</tr>
 
</tr>
 
</table>
 
</table>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
  
 +
<div class="column half_size">
 +
<div align="center">
 +
 +
<h4> Phosphatase of linearized pSBK3 </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Amount</th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Digestion of plasmid </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<td> Phosphatase </td>
 +
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 7 </td>
 +
</tr>
 +
</table>
 +
</ul>
 
</div>
 
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
 +
<h4> Ligate into Plasmid treated with Phosphatase </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Amount </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> Plasmid </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> Toggle pieces </td>
 +
<td> 10 (500 ng/uL) </td>
 +
</tr>
 +
<td> Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<h5>
 +
Transformation of cells with toggle pieces
 +
</h5>
 +
<ul>
 +
<li>
 +
5 uL of each DNA solution
 +
</li>
 +
 +
 +
 +
 +
  
  
Line 506: Line 583:
 
</div>
 
</div>
  
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Incubation </h4>
 +
<ul>
 +
<li> Incubated overnight </li>
 +
</ul>
 +
</div>
 +
</div>
  
  
Line 518: Line 603:
 
<div align="center">
 
<div align="center">
 
<h2 > 9/1 </h2>
 
<h2 > 9/1 </h2>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
 +
<h4> Deactivation of Digest with Phosphatase </h4>
 +
 +
<br>
 +
<br>
 +
 +
<h4> PCR of Toggle #1 </h4>
 +
<ul>
 +
<li> Same setup as 8/30 </li>
 +
</ul>
 +
 +
<br>
 +
 +
<h4> Ligation of CR-1 into pEP352GAP </h4>
 
</div>
 
</div>
 
</div>
 
</div>
Line 530: Line 634:
 
<div align="center">
 
<div align="center">
 
<h2 > 9/2 </h2>
 
<h2 > 9/2 </h2>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> PCR of Toggle 1 </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Phire </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 8.0 </td>
 +
</tr>
 +
<tr>
 +
<td> 5 uM F Primer </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
<tr>
 +
<td> 5 uM R Primer </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
<tr>
 +
<td> 100pg/ul DNA </td>
 +
<td> 1.0 </td>
 +
</tr>
 +
</table>
 +
<ul>
 +
<li> Also ran trials with 1.5, 2.5, 3.5 and 4.5 ul of both forward and reverse primers </li>
 +
<li> Adjusted H<sub>2</sub>O accordingly </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> PCR Settings: 30 cycles </h5>
 +
<table>
 +
<tr>
 +
<th> Phase </th>
 +
<th> Temperature (°C) </th>
 +
<th> Time (sec)</th>
 +
</tr>
 +
<tr>
 +
<td> Initial Denaturation </td>
 +
<td> 98 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 67 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 25 </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60 </td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Another PCR of Toggle 1 </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Phire </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 7.74 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 0.63 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 0.63 </td>
 +
</tr>
 +
<tr>
 +
<td> 100pg/ul DNA </td>
 +
<td> 1.0 </td>
 +
</tr>
 +
</table>
 +
<ul>
 +
<li> Ran trials with 1.25 and 1.88 ul of forward and reverse primers </li>
 +
<li> H<sub>2</sub>O for those trials were 6.5 and 5.24 ul respectively </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> PCR Settings: 35 cycles </h5>
 +
<table>
 +
<tr>
 +
<th> Phase </th>
 +
<th> Temperature (°C) </th>
 +
<th> Time (sec)</th>
 +
</tr>
 +
<tr>
 +
<td> Initial Denaturation </td>
 +
<td> 98 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 68.5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 20 </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60 </td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> PCR of Toggle 2 </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Phire </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 7.74 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 0.63 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 0.63 </td>
 +
</tr>
 +
<tr>
 +
<td> 100pg/ul DNA </td>
 +
<td> 1.0 </td>
 +
</tr>
 +
</table>
 +
<ul>
 +
<li> Ran trials with 1.25 and 1.88 ul of forward and reverse primers </li>
 +
<li> H<sub>2</sub>O for those trials were 6.5 and 5.24 ul respectively </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> PCR Settings: 35 cycles </h5>
 +
<table>
 +
<tr>
 +
<th> Phase </th>
 +
<th> Temperature (°C) </th>
 +
<th> Time (sec)</th>
 +
</tr>
 +
<tr>
 +
<td> Initial Denaturation </td>
 +
<td> 98 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 10 </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 68.5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 35 </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60 </td>
 +
</tr>
 +
</table>
 
</div>
 
</div>
 
</div>
 
</div>

Latest revision as of 03:38, 7 September 2016

Week 10: 8/29 - 9/4

Week 10: 8/29 - 9/4



8/29

AmilCP BioBrick Digests

Content Experimental ul Control ul
H2O 46.5 47.0
Cutsmart 5.0 5.0
XbaI 0.5 X
DNA 2.0 2.0

PCR of Toggle 1

Contents ul
Phire 10
H2O 8
5 uM F Primer 1.5
5 uM R Primer 1.5
100pg/ul Construct 1.0

  • Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
  • Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 5
Annealing 68 5
Extension 72 45
Final Extension 72 60

PCR of Toggle 2

Contents ul
Phire 10
H2O 8
5 uM F Primer 1.5
5 uM R Primer 1.5
100pg/ul Construct 1.0

  • Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
  • Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 5
Annealing 68 5
Extension 72 45
Final Extension 72 60

Digests of Toggle parts

Toggle 1 Digest
Content Experimental ul Control ul
H2O 24 25
Cutsmart 5.0 5.0
SpeI 1.0 X
DNA 25.0 25.0
Toggle 1 Digest
Content Experimental ul Control ul
H2O 24 25
Cutsmart 5.0 5.0
XbaI 1.0 X
DNA 25.0 25.0

Linearized pSB1C7 Digest


Content Experimental ul Control ul
H2O 16.5 18.5
Cutsmart 2.5 2.5
SpeI-HF 1.0 X
XbaI 1.0 X
DNA 4.0 4.0


Ligation of Toggle parts

Content ul
10X T4 Buffer 2.0
Toggle 1 5.0
Toggle 2 10.0
H2O 2.0
Ligase 1.0


8/30

Ligate Toggle 2 into Plasmid

    Content ul
    Ligase Buffer 2
    Toggle pieces 10 (500 ng/ul)
    Plasmid 2
    H2O 5
    Ligase 1

Phosphatase of linearized pSBK3

    Amount ul
    Digestion of plasmid 10
    Buffer 2
    Phosphatase 1
    H2O 7

Ligate into Plasmid treated with Phosphatase

    Amount ul
    Plasmid 5
    Toggle pieces 10 (500 ng/uL)
    Buffer 2
    H2O 2
    Ligase 1
Transformation of cells with toggle pieces
  • 5 uL of each DNA solution


    • 8/31

    Digest of IDT CR-1 SBU and Vector

    CR-1 SBU
    Content Experimental ul Control ul
    H2O 18 20
    Cutsmart 5.0 5.0
    EcoRI-HF 1.0 X
    XhoI 1.0 X
    DNA 25.0 25.0
pEP352GAP
Content Experimental ul Control ul
H2O 7.0 8.0
Cutsmart 1.0 1.0
EcoRI-HF 0.5 X
XhoI 0.5 X
DNA 1.0 1.0

Incubation

  • Incubated overnight


9/1

Deactivation of Digest with Phosphatase



PCR of Toggle #1

  • Same setup as 8/30

Ligation of CR-1 into pEP352GAP



9/2

PCR of Toggle 1

Contents ul
Phire 10
H2O 8.0
5 uM F Primer 0.5
5 uM R Primer 0.5
100pg/ul DNA 1.0
  • Also ran trials with 1.5, 2.5, 3.5 and 4.5 ul of both forward and reverse primers
  • Adjusted H2O accordingly
PCR Settings: 30 cycles
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 10
Annealing 67 5
Extension 72 25
Final Extension 72 60

Another PCR of Toggle 1

Contents ul
Phire 10
H2O 7.74
10 uM F Primer 0.63
10 uM R Primer 0.63
100pg/ul DNA 1.0
  • Ran trials with 1.25 and 1.88 ul of forward and reverse primers
  • H2O for those trials were 6.5 and 5.24 ul respectively
PCR Settings: 35 cycles
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 10
Annealing 68.5 5
Extension 72 20
Final Extension 72 60

PCR of Toggle 2

Contents ul
Phire 10
H2O 7.74
10 uM F Primer 0.63
10 uM R Primer 0.63
100pg/ul DNA 1.0
  • Ran trials with 1.25 and 1.88 ul of forward and reverse primers
  • H2O for those trials were 6.5 and 5.24 ul respectively
PCR Settings: 35 cycles
Phase Temperature (°C) Time (sec)
Initial Denaturation 98 30
Denaturation 98 10
Annealing 68.5 5
Extension 72 35
Final Extension 72 60


9/3



9/4