Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W9"

Line 171: Line 171:
 
</tr>
 
</tr>
 
</table>
 
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="coloumn full_size">
 +
<div align="center">
 +
<h4> Digest of BioBricks </h4>
 +
<br>
 +
<br>
 +
<h5> J23119 - 225.8ng</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> EcoRI-HF </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> SpeI</td>
 +
<td> 0.5</td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 44.3 </td>
 +
<td> 2.2</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> B0015 - 201.5ng</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> XbaI </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> PstI</td>
 +
<td> 0.5</td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 49.6 </td>
 +
<td> 2.5</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> E0040 - 179.3ng</h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Experimental ul </th>
 +
<th> Control ul </th>
 +
</tr>
 +
<tr>
 +
<td> Cutsmart </td>
 +
<td> 5 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> EcoRI-HF </td>
 +
<td> 0.5 </td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> SpeI</td>
 +
<td> 0.5</td>
 +
<td> X </td>
 +
</tr>
 +
<tr>
 +
<td> DNA </td>
 +
<td> 55.8 </td>
 +
<td> 2.8</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> LB Prep </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5>J23119</h5>
 +
<ul>
 +
<li> 8ml LB</li>
 +
<li> 2ml chloramphenicol</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5>K592009</h5>
 +
<ul>
 +
<li> 8ml LB</li>
 +
<li> 2ml chloramphenicol</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5>E0040</h5>
 +
<ul>
 +
<li> 8ml LB</li>
 +
<li> 2ml Ampicillin</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5>B0015</h5>
 +
<ul>
 +
<li> 8ml LB</li>
 +
<li> 2ml chloramphenicol</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 184: Line 332:
 
<div align="center">
 
<div align="center">
 
<h2> 8/23 </h2>
 
<h2> 8/23 </h2>
 +
</div>
 +
</div>
 +
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> PCR of amilCP, #1 and #2 with 67° annealing temperature </h4>
 +
<ul>
 +
<li> Ran a gel afterwards </li>
 +
</ul>
 +
<br>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> AmilCP </li>
 +
<li> #1 </li>
 +
<li> #2 </li>
 +
</ol>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> Gel of 10ug overnight BB digest </h4>
 +
<h6> Lanes:</h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> J23119 </li>
 +
<li> J23119 Control </li>
 +
<li> B0015 </li>
 +
<li> B0015 Control </li>
 +
<li> E0040 </li>
 +
<li> E0040 Control </li>
 +
</ol>
 
</div>
 
</div>
 
</div>
 
</div>
Line 189: Line 371:
 
<div class="column full_size">
 
<div class="column full_size">
 
<div align="center">
 
<div align="center">
 +
<h4> Competent Cells (TSS) </h4>
 +
<h5> 50ml TSS </h5>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Amount</th>
 +
</tr>
 +
<tr>
 +
<td> PEG3350 </td>
 +
<td> 5g </td>
 +
</tr>
 +
<tr>
 +
<td> 1M MgCL<sub>2</sub> </td>
 +
<td> 1.5 ml </td>
 +
</tr>
 +
<td> DMSO </td>
 +
<td> 2.5ml </td>
 +
</tr>
 +
<tr>
 +
<td> LB </td>
 +
<td> Fill to 50ml </td>
 +
</tr>
 +
</table>
 +
<ul>
 +
<li> Tested pH to be ~6.5</li>
 +
</ul>
 +
<br>
 
</div>
 
</div>
 
</div>
 
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> New PCR of Toggle #1 and amil CP </h4>
 +
<ul>
 +
<li> 69°C annealing temperature </li>
 +
<li> Extension length - 2.5 mins </li>
 +
<li> Ran for 30 Cycles </li>
 +
</ul>
 +
<br>
 +
<h4> Ran a gel </h4>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> amil CP </li>
 +
<li> #4 </li>
 +
<li> Ladder </li>
 +
<ol>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> Gel of Digested BB Parts </h4>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> J23119 #1</li>
 +
<li> J23119 #2</li>
 +
<li> B0015 #1 </li>
 +
<li> B0015 #2 </li>
 +
<li> E0040 #1 </li>
 +
<li> E0040 #2 </li>
 +
</ol>
 +
</div>
 +
</div>
 +
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Nanodrops of BioBricks and PCR purifications </h4>
 +
<table>
 +
<tr>
 +
<th> Content </th>
 +
<th> Concentration (ng/ul) </th>
 +
<th> 260/280 </th>
 +
</tr>
 +
<tr>
 +
<td> R592009 </td>
 +
<td> 234.3</td>
 +
<td> 1.92 </td>
 +
</tr>
 +
<tr>
 +
<td> J23119 </td>
 +
<td> 93.0 </td>
 +
<td> 1.95 </td>
 +
</tr>
 +
<tr>
 +
<td> B0015 </td>
 +
<td> 229.4 </td>
 +
<td> 2.00 </td>
 +
</tr>
 +
<tr>
 +
<td> E0040 </td>
 +
<td> 287.5 </td>
 +
<td> 1.95 </td>
 +
</tr>
 +
<tr>
 +
<td> PiggyBac </td>
 +
<td> 274.0 </td>
 +
<td> 1.89 </td>
 +
</tr>
 +
<tr>
 +
<td> Toggle #2 </td>
 +
<td> 9.6 </td>
 +
<td> 2.45 </td>
 +
</tr>
 +
</table>
 +
<br>
 +
 +
<h4> Digests </h4>
 +
<ul>
 +
<li> Re-do of the the digest from 8/21 </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> PCR of Amil CP and #1</h4>
 +
<ul>
 +
<li> 67°C annealing temperature for 15 seconds </li>
 +
<li> 60 second extension </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> 25ul PCR of Toggle #2 </h4>
 +
<ul>
 +
<li> 68°C annealing temperature</li>
 +
<li> Primer concentrations at 100-500 ng/ul</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Gel of BioBrick Digests </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> Gel #1 </h5>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> B0015 </li>
 +
<li> B0015 </li>
 +
<li> B0015 Control </li>
 +
<li> J12119 </li>
 +
<li> J23119 </li>
 +
<li> J23119 Control </li>
 +
</ol>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h5> Gel #2 </h5>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> K592009 </li>
 +
<li> K592009</li>
 +
<li> K592009 </li>
 +
<li> K592009 Control </li>
 +
<li> E0040</li>
 +
<li> E0040 </li>
 +
<li> E0040 Control </li>
 +
</ol>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> PCR of Toggle #2 - 30x </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Q5 MasterMix </td>
 +
<td> 12.5 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 5.5</td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 1.0 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 1.0 </td>
 +
</tr>
 +
<tr>
 +
<td> Template</td>
 +
<td> 5 </td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Q5 MasterMix </td>
 +
<td> 12.5 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 3.5</td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 2.0 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 2.0 </td>
 +
</tr>
 +
<tr>
 +
<td> Template</td>
 +
<td> 5 </td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Gel </h4>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> Toggle #2 - 1ul primer </li>
 +
<li> Toggle #2 - 2ul primer </li>
 +
</ol>
 +
<br>
 +
</div>
 +
</div>
 +
 +
<div class="column full_size">
 +
<div align="center">
 +
<h4> Toggle #1 Trials </h4>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> PCR of Toggle #1 </h4>
 +
<ul>
 +
<li> Ran 6 trials. The first started with 0.5 ul of each of the F and R primers.</li>
 +
<li> Each trial increased by 0.5 until we got to 2.5ul of primers</li>
 +
<li> Ran a 0.25ul primer trial </li>
 +
<li> Used 12.5ul of Q5 MasterMix and 5ul of template </li>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<h4> Toggle #1 PCR </h4>
 +
<h6> Lanes: </h6>
 +
<ol>
 +
<li> Ladder </li>
 +
<li> 0.25 </li>
 +
<li> 0.5 </li>
 +
<li> 1.0 </li>
 +
<li> 1.5 </li>
 +
<li> 2.0 </li>
 +
<li> 2.5 </li>
 +
</ol>
 +
</div>
 +
</div>
 +
  
 
<div align="center">
 
<div align="center">

Revision as of 19:56, 25 August 2016

Week 9: 8/22 - 8/28

Week 9: 8/22 - 8/28



8/22

PCR of new IDT Shipments

AmilCP
    Contents ul
    Q5 MasterMix 12.5
    H2O 9
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Template 1
IDT #1
    Contents ul
    Q5 MasterMix 12.5
    H2O 6.5
    10 uM F Primer 2.5
    10 uM R Primer 2.5
    Template 1

IDT #2
    Contents ul
    Q5 MasterMix 12.5
    H2O 6.5
    10 uM F Primer 2.5
    10 uM R Primer 2.5
    Template 1

PCR Settings: 25 Cycles

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30
    Denaturation 98 10
    Annealing 65 30
    Extension 72 120
    Final Extension 72 120

Digest of BioBricks



J23119 - 225.8ng
Content Experimental ul Control ul
Cutsmart 5 5
EcoRI-HF 0.5 X
SpeI 0.5 X
DNA 44.3 2.2
B0015 - 201.5ng
Content Experimental ul Control ul
Cutsmart 5 5
XbaI 0.5 X
PstI 0.5 X
DNA 49.6 2.5
E0040 - 179.3ng
Content Experimental ul Control ul
Cutsmart 5 5
EcoRI-HF 0.5 X
SpeI 0.5 X
DNA 55.8 2.8

LB Prep

J23119
  • 8ml LB
  • 2ml chloramphenicol
K592009
  • 8ml LB
  • 2ml chloramphenicol
E0040
  • 8ml LB
  • 2ml Ampicillin
B0015
  • 8ml LB
  • 2ml chloramphenicol


8/23

PCR of amilCP, #1 and #2 with 67° annealing temperature

  • Ran a gel afterwards

Lanes:
  1. Ladder
  2. AmilCP
  3. #1
  4. #2

Gel of 10ug overnight BB digest

Lanes:
  1. Ladder
  2. J23119
  3. J23119 Control
  4. B0015
  5. B0015 Control
  6. E0040
  7. E0040 Control

Competent Cells (TSS)

50ml TSS
Content Amount
PEG3350 5g
1M MgCL2 1.5 ml
DMSO 2.5ml
LB Fill to 50ml
  • Tested pH to be ~6.5

New PCR of Toggle #1 and amil CP

  • 69°C annealing temperature
  • Extension length - 2.5 mins
  • Ran for 30 Cycles

Ran a gel

Lanes:
  1. amil CP
  2. #4
  3. Ladder

Gel of Digested BB Parts

Lanes:
  1. Ladder
  2. J23119 #1
  3. J23119 #2
  4. B0015 #1
  5. B0015 #2
  6. E0040 #1
  7. E0040 #2

Nanodrops of BioBricks and PCR purifications

Content Concentration (ng/ul) 260/280
R592009 234.3 1.92
J23119 93.0 1.95
B0015 229.4 2.00
E0040 287.5 1.95
PiggyBac 274.0 1.89
Toggle #2 9.6 2.45

Digests

  • Re-do of the the digest from 8/21

PCR of Amil CP and #1

  • 67°C annealing temperature for 15 seconds
  • 60 second extension

25ul PCR of Toggle #2

  • 68°C annealing temperature
  • Primer concentrations at 100-500 ng/ul

Gel of BioBrick Digests

Gel #1
Lanes:
  1. Ladder
  2. B0015
  3. B0015
  4. B0015 Control
  5. J12119
  6. J23119
  7. J23119 Control
Gel #2
Lanes:
  1. Ladder
  2. K592009
  3. K592009
  4. K592009
  5. K592009 Control
  6. E0040
  7. E0040
  8. E0040 Control

PCR of Toggle #2 - 30x

Contents ul
Q5 MasterMix 12.5
H2O 5.5
10 uM F Primer 1.0
10 uM R Primer 1.0
Template 5
Contents ul
Q5 MasterMix 12.5
H2O 3.5
10 uM F Primer 2.0
10 uM R Primer 2.0
Template 5

Gel

Lanes:
  1. Ladder
  2. Toggle #2 - 1ul primer
  3. Toggle #2 - 2ul primer

Toggle #1 Trials

PCR of Toggle #1

  • Ran 6 trials. The first started with 0.5 ul of each of the F and R primers.
  • Each trial increased by 0.5 until we got to 2.5ul of primers
  • Ran a 0.25ul primer trial
  • Used 12.5ul of Q5 MasterMix and 5ul of template

Toggle #1 PCR

Lanes:
  1. Ladder
  2. 0.25
  3. 0.5
  4. 1.0
  5. 1.5
  6. 2.0
  7. 2.5


8/24