Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W14"

Line 130: Line 130:
 
<div align="center">
 
<div align="center">
 
<h2 > 10/1 </h2>
 
<h2 > 10/1 </h2>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Constructs of T1 and T2 </h4>
 +
<ul>
 +
<li> Ran a gel of the constructs</li>
 +
<li> Set up tubes for TSS Competent cells </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> This </h5>
 +
</div>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O </td>
 +
<td> #</td>
 +
<td> # </td>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> # </td>
 +
<td> # </td>
 +
</tr>
 +
<tr>
 +
<td> EcoRI-HF </td>
 +
<td> # </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> XbaI </td>
 +
<td># </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> # </td>
 +
<td> # </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> CRISBU </h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> EcoRI-HF </td>
 +
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> XhoI </td>
 +
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 43 </td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> Toggle 1</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> EcoRI-HF </td>
 +
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> SpeI-HF </td>
 +
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 28 </td>
 +
</tr>
 +
</table>
 +
<ul>
 +
<li> Filled to 50ul with water</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h5> Toggle 2 </h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> EcoRI-HF </td>
 +
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> PstI-HF </td>
 +
<td> 1 </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 35 </td>
 +
</tr>
 +
</table>
 +
<ul>
 +
<li> Filled to 50ul with water</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<h4> Ligations </h4>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pEPGAP</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 2 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 0 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 15 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pEPGAP/CRISBU</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 2 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 2</td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 13 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pEPGAP-P </h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 4 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 0 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 13 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pEPGAP-P/CRISBU</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 4 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 4 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 8 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pSBIA2/T1/T2</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 1 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 13.52 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 2.5 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pSBIA2</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 1 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 0 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 16 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pSBIA2-p</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 2 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 0 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 15 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> pSBIA2-P/T1/T2</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> ul </th>
 +
</tr>
 +
<tr>
 +
<td> 10X T4 Buffer </td>
 +
<td> 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Vector </td>
 +
<td> 2 </td>
 +
</tr>
 +
<td> Insert </td>
 +
<td> 13.50 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 2.5 </td>
 +
</tr>
 +
<tr>
 +
<td> Ligase </td>
 +
<td> 1</td>
 +
</tr>
 +
</table>
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 02:53, 12 October 2016

Week 14: 9/26 - 10/2

Week 14: 9/26 - 10/2



9/26



9/27



9/28



9/29

Toggle 2 Gel Construct using Phire Polymerase



PCR Ran of Toggle 2:
    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 60s
    Denaturation 98 20s
    Annealing Ranged from 72-60 degrees for each tube. Did a different annealing temperature for each PCR. 10s
    Extension 72 #
    Final Extension 72 #
    Ran 32 cycles


9/30



10/1

Constructs of T1 and T2

  • Ran a gel of the constructs
  • Set up tubes for TSS Competent cells
This
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #
CRISBU
Content Experimental ul
Cutsmart 5
EcoRI-HF 1
XhoI 1
DNA 43
Toggle 1
Content Experimental ul
Cutsmart 5
EcoRI-HF 1
SpeI-HF 1
DNA 28
  • Filled to 50ul with water
Toggle 2
Content Experimental ul
Cutsmart 5
EcoRI-HF 1
PstI-HF 1
DNA 35
  • Filled to 50ul with water

Ligations

pEPGAP
Content ul
10X T4 Buffer 2
Vector 2
Insert 0
H2O 15
Ligase 1
pEPGAP/CRISBU
Content ul
10X T4 Buffer 2
Vector 2
Insert 2
H2O 13
Ligase 1
pEPGAP-P
Content ul
10X T4 Buffer 2
Vector 4
Insert 0
H2O 13
Ligase 1
pEPGAP-P/CRISBU
Content ul
10X T4 Buffer 2
Vector 4
Insert 4
H2O 8
Ligase 1
pSBIA2/T1/T2
Content ul
10X T4 Buffer 2
Vector 1
Insert 13.52
H2O 2.5
Ligase 1
pSBIA2
Content ul
10X T4 Buffer 2
Vector 1
Insert 0
H2O 16
Ligase 1
pSBIA2-p
Content ul
10X T4 Buffer 2
Vector 2
Insert 0
H2O 15
Ligase 1
pSBIA2-P/T1/T2
Content ul
10X T4 Buffer 2
Vector 2
Insert 13.50
H2O 2.5
Ligase 1


10/2

Transformations

  1. Thaw TSS cells on ice
  2. Add 2ul of prepped plasmid. Pipette to mix
  3. Sit for 30 minutes on ice
  4. Incubate cells for 30s at 43°C
  5. Incubate on ice for 2 minutes
  6. Add 1ml of 50C
  7. Incubate for 30 minutes at 37°C. Shake
  8. Spread 100-300ul on plates
  9. Grow overnight at 37°C
  10. Save the rest as liquid cultures

Plates Used:

  • Plate 1: pEPGAP
  • Plate 2: pEPGAP/CRISBU
  • Plate 3: pEPGAP-P
  • Plate 4:
  • Plate 5: pSBIA2/T1/T2
  • Plate 6: pSBIA2
  • Plate 7: pSBIA2-P
  • Plate 8: