Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W6"

Line 398: Line 398:
 
<ul>
 
<ul>
 
<li> Did not work </li>
 
<li> Did not work </li>
 +
</ul>
 +
 +
<h5>PCR of miniprepped project plasmids to determine ligation</h5>
 +
 +
<h5> PCR Contents: </h5>
 +
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Phire </td>
 +
<td> 10 uL </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 7.5 uL </td>
 +
</tr>
 +
<tr>
 +
<td> 50 uM F Primer </td>
 +
<td> 1 uL </td>
 +
</tr>
 +
<tr>
 +
<td> 50 uM R Primer </td>
 +
<td> 1 uL </td>
 +
</tr>
 +
<tr>
 +
<td> Plasmids: 366.6 ng/uL, 301 ng/uL, 259 ng/uL </td>
 +
<td> 0.5 uL </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
 +
<h4> PCR Settings: </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> 30s </td>
 +
</tr>
 +
<tr>
 +
<td> Denaturation </td>
 +
<td> 98 </td>
 +
<td> 20s </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 66 </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 30s </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60s </td>
 +
</tr>
 +
</table>
 
</ul>
 
</ul>

Revision as of 15:00, 8 August 2016

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Week 6: 8/1-8/5

8/1-8/9

8/1

Emmanuel Ligation (193 ng/ul)

    Content Experimental (ul) Control (ul)
    H2O 38.8 39.8
    Cutsmart 5.0 5.0
    XbaI 0.5 X
    EcoRI-HF 0.5 X
    DNA 5.18 5.18

Gel run

Lanes:

  1. Ladder
  2. Emmanuel Digest Control
  3. Emmanuel Digest Experimental
  • Did not work

25 ul Digest of Ryan + Emmanuel (Veronica) Miniprep

  • Made 17 LB With Ampicillin (600ml)
Ryan - 446.4 ng/ul
    Content Experimental (ul) Control (ul)
    H2O 20.4 21.4
    Cutsmart 2.5 2.5
    XbaI 0.5 X
    EcoRI-HF 0.5 X
    DNA 1.12 1.12
Emmanuel - 193 ng/ul
    Content Experimental (ul) Control (ul)
    H2O 18.9 19.9
    Cutsmart 2.5 2.5
    XbaI 0.5 X
    EcoRI-HF 0.5 X
    DNA 2.59 2.59

8/2

Miniprep

  • Miniprep of ligated construct
  • 10,000rpm for 3 minutes at 25°C

Ran a nanodrop of Minipreps

    Content Concentration (ng/ul) 260/280
    Veronica 1 311.5 1.86
    Veronica 2 43.8 1.76
    Veronica 3 259 1.88
    Veronica 4 296 1.88
    R1 176.5 1.75
    Ryan 2 277.3 1.85
    Ryan 3 301.6 1.81
    Ryan 4 366.6 1.82

Ran a gel of miniprepped plasmids

  • Used 5ul of DNA and 1 ul of dye

Lanes:

  1. Veronica 1
  2. Veronica 3
  3. Veronica 4
  4. Ladder
  5. Ryan 1
  6. Ryan 2
  7. Ryan 3
  8. Ryan 4

Digest of Miniprepped Plasmids

Veronica 1 - 311.5 ng/ul
    Content Experimental (ul) Control (ul)
    H2O 40.8 41.8
    Cutsmart 5 5
    XbaI 0.5 X
    PvuII-HF 0.5 X
    DNA 3.21 3.21
Ryan 4 - 366.6 ng/ul
    Content Experimental (ul) Control (ul)
    H2O 41.3 42.3
    Cutsmart 5 5
    XbaI 0.5 X
    PvuII-HF 0.5 X
    DNA 2.73 2.73

8/3

Transformations

  • Transformed R001, J61100, puC19
  • 500 uL of SOC used for each transformation

Miniprep of BioBricks from 8/2

Gel run ( 6uL of each tube loaded)

Lanes:

  1. 5 uL plasmid
  2. 5 uL plasmid + 5 uL of nuclease free water
  3. 5 uL plasmid + 5 uL water + 2.5 uL of CutSmart Buffer
  4. Ladder
  5. 5 uL plasmid + 5 uL water + 2.5 uL CutSmart + 0.75 uL XhoI
  6. 5 ul plasmid + 5 uL water + 2.5 uL CutSmart + 0.75 uL PvuII
  7. 5 ul plasmid + 6 uL water + 2.5 uL CutSmart + 0.75 uL XhoI + 0.75 uL PvuII
  • Did not work

Interlab LB cultures made from plates

Developed sequencing primers for construct

8/4

Worked on InterLab study

Gel ran (done by Emmanuel)

Lanes:

  1. Veronica control (plasmid)
  2. Veronica digest
  3. Ladder
  4. Ryan digest
  5. Ryan control (plasmid)
  • Did not work
PCR of miniprepped project plasmids to determine ligation
PCR Contents:
    Contents ul
    Phire 10 uL
    H2O 7.5 uL
    50 uM F Primer 1 uL
    50 uM R Primer 1 uL
    Plasmids: 366.6 ng/uL, 301 ng/uL, 259 ng/uL 0.5 uL

PCR Settings:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30s
    Denaturation 98 20s
    Annealing 66 5
    Extension 72 30s
    Final Extension 72 60s