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

 
(14 intermediate revisions by 2 users not shown)
Line 17: Line 17:
 
<br>
 
<br>
 
</div>
 
</div>
 +
 +
  
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h2 > 9/26 </h2>
+
<h2 > 9/29 </h2>
 
</div>
 
</div>
 
</div>
 
</div>
 +
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Toggle 2 Gel Construct using Phire Polymerase </h4>
  
 
<div align="center">
 
<div align="center">
 
<br>
 
<br>
−
<hr>
 
 
<br>
 
<br>
 
</div>
 
</div>
  
−
<div class="column full_size">
+
<h5> PCR Ran of Toggle 2: </h5>
−
<div align="center">
+
<ul>
−
<h2 > 9/27 </h2>
+
<table>
 +
<tr>
 +
<th> Phase </th>
 +
<th> Temperature (°C) </th>
 +
<th> Time (sec)</th>
 +
</tr>
 +
<tr>
 +
<td> Initial Denaturation </td>
 +
<td> 98 </td>
 +
<td> 60s </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 20s </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> Ranged from 72-60 degrees for each tube. Did a different annealing temperature for each PCR. </td>
 +
<td> 10s </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> # </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> # </td>
 +
</tr>
 +
</table>
 +
<h6> Ran 32 cycles </h6>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 44: Line 83:
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h2 > 9/28 </h2>
+
<h2 > 9/30 </h2>
 +
</div>
 +
</div>
 +
 
 +
<div class="column full_size">
 +
<div align="left">
 +
<ul>
 +
<li> Ran PCR of CRISBu </li>
 +
<li> Ligation of CrisBu into pEP352GAP </li>
 +
<li> Nanodrop and PCR of Toggle 2 </li>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> PCR Toggle 2: </h5>
 +
 
 +
<ul>
 +
<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, 6, and 4 for three tubes respectively</td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 0.5, 1.0, and 2.0 for three tubes respectively</td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 0.5, 1.0, and 2.0 for three tubes respectively </td>
 +
</tr>
 +
<tr>
 +
<td> 100ug/ul Construct </td>
 +
<td> 2 uL</td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> PCR Settings Toggle 2: </h4>
 +
 
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Phase </th>
 +
<th> Temperature (°C) </th>
 +
<th> Time (sec)</th>
 +
</tr>
 +
<tr>
 +
<td> Initial Denaturation </td>
 +
<td> 98 </td>
 +
<td> 60s </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 10s </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 69 </td>
 +
<td> 10s </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 40s </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60s </td>
 +
</tr>
 +
</table>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 56: Line 178:
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h2 > 9/29 </h2>
+
<h2 > 10/1 </h2>
 
</div>
 
</div>
 
</div>
 
</div>
  
 +
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<br>
+
<h4> Constructs of T1 and T2 </h4>
−
<hr>
+
<ul>
−
<br>
+
<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> 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>
  
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<h2 > 9/30 </h2>
+
<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>
 
</div>
  
 +
<div class="column full_size">
 
<div align="center">
 
<div align="center">
−
<br>
+
<h4> Ligations </h4>
−
<hr>
+
</div>
−
<br>
+
</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>
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
</div>
 +
 +
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h2 > 10/1 </h2>
+
<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>
Line 113: Line 575:
 
<br>
 
<br>
  
 +
<h4> Plates Used: </h4>
 
<ul>
 
<ul>
 
<li> Plate 1: pEPGAP </li>
 
<li> Plate 1: pEPGAP </li>

Latest revision as of 07:54, 19 October 2016

Week 14: 9/26 - 10/2

Week 14: 9/26 - 10/2



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

  • Ran PCR of CRISBu
  • Ligation of CrisBu into pEP352GAP
  • Nanodrop and PCR of Toggle 2
PCR Toggle 2:
    Contents ul
    Phire 10
    H2O 7, 6, and 4 for three tubes respectively
    10 uM F Primer 0.5, 1.0, and 2.0 for three tubes respectively
    10 uM R Primer 0.5, 1.0, and 2.0 for three tubes respectively
    100ug/ul Construct 2 uL

PCR Settings Toggle 2:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 60s
    Denaturation 98 10s
    Annealing 69 10s
    Extension 72 40s
    Final Extension 72 60s


10/1

Constructs of T1 and T2

  • Ran a gel of the constructs
  • Set up tubes for TSS Competent cells
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: