Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W7"

Line 563: Line 563:
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<div align="center">
 +
<h2> 8/10 </h2>
 +
</div>
 +
<h4> 25 ul PCR of Miniprepped Plasmids for Project</h4>
 +
</div>
 +
 +
<div class="column half_size">
 +
<div align="center">
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Q5 Buffer </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> Q5 Polymerase </td>
 +
<td> 5 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 16.25 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 1.25 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 1.25 </td>
 +
</tr>
 +
<tr>
 +
<td> Construct </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
</div>
 +
</div>
 +
 +
<div class="column half_size">
 
<div align="center">
 
<div align="center">
−
<h2> 8/10 </h2>
+
<ul>
 +
<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> 20 </td>
 +
</tr>
 +
<tr>
 +
<td> Annealing </td>
 +
<td> 56</td>
 +
<td> 15 </td>
 +
</tr>
 +
<tr>
 +
<td> Extension </td>
 +
<td> 72 </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> Final Extension </td>
 +
<td> 72 </td>
 +
<td> 60 </td>
 +
</tr>
 +
</table>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
 +
 +
<div class="column full_size">
 +
<h5> PCR was run on the following construct concentrations in ng/ul</h5>
 +
<ul>
 +
<li> 273</li>
 +
<li>176.5</li>
 +
<li>296.6</li>
 +
<li>311.5</li>
 +
<li>301.6</li>
 +
<li>301.6</li>
 +
<li> 259</li>
 +
<li> 366.6</li>
 +
</ul>
 +
<ul>
 +
<li> PCRs did not work → Changed annealing temperature to 66°C</li>
 +
<li> Re-ran the first 5 constructs on the above list </li>
 +
</ul>
 +
 +
<h4> 25 ul PCR of Miniprepped Plasmid for BioBrick Portion </h4>
 +
<ul>
 +
<table>
 +
<tr>
 +
<th> Contents </th>
 +
<th> ul </th>
 +
</tr>
 +
<td> Q5 Master Mix </td>
 +
<td> 12.5 </td>
 +
</tr>
 +
<tr>
 +
<td> H<sub>2</sub>O</td>
 +
<td> 9.5 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM F Primer </td>
 +
<td> 1.25 </td>
 +
</tr>
 +
<tr>
 +
<td> 10 uM R Primer </td>
 +
<td> 1.25 </td>
 +
</tr>
 +
<tr>
 +
<td> Template </td>
 +
<td> 0.5 </td>
 +
</tr>
 +
</table>
 +
</ul>
 +
 +
<ul>
 +
<li> This setup was used for all BioBrick Parts </li>
 +
<li> Ran with the "Sunil" Program with a 66°C annealing temperature</li>
  
 
<div class="column full_size">
 
<div class="column full_size">

Revision as of 15:48, 15 August 2016

MENU ▤

Week 7: 8/8 - 8/14

I left you some of the commonly used templates down there under 8/8. Just fill them in as needed. I'll only really have wifi for a few hours on two days between Saturday and Thursday. If you need anything, message me on Facebook and I'll get back to you as soon as I can.

With the warmest love and regards,

~JOSHUA

Week 7: 8/8 - 8/14

PCR Table Template:
    Contents ul
    Phire #
    H2O #
    50 uM F Primer #
    50 uM R Primer #
    #ug/ul Construct #

PCR Settings Template:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 #
    Denaturation 98 #
    Annealing # #
    Extension 72 #
    Final Extension 72 #

Nanodrop Template:

    Content Concentration (ng/ul) 260/280
    Construct # #
    Vector # #

Ligation Template

    Content ul
    10X T4 Buffer #
    Vector # (# ng)
    Construct # (# ng)
    H2O #
    Ligase #

Multiple Digests Template:

This
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #
Is
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #

A
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #
Thing
    Content Experimental ul Control ul
    H2O # #
    Cutsmart # #
    EcoRI-HF # X
    XbaI # X
    DNA # #

8/8

Sequencing

    T1 T2
    5.34 uL H2O 5.34 uL H2O
    1 uL CR-1 Primer 1 1 uL CR-1 Primer 2
    1.66 uL DNA (301.6 ng/uL) 1.66 uL DNA (301.6 ng/uL)

Diluted BioBrick MiniPreps for PCR

    BioBrick Name Concentration (ng/uL) Contents (DNA + water)
    K592009 76.9 1 uL + 6.69 uL water
    B0015 51.7 1 uL + 4.17 uL water
    K1033910 156.5 1 uL + 14.65 uL water
    R0082 49.5 1 uL + 3.95 uL water
    C0012 150.8 1 uL + 14.08 uL water
    J61100 123.0 1 uL + 11.3 uL water
    R0011 56.7 1 uL + 4.67 uL water

PCR of each BioBrick parts (25 uL Q5 MasterMix rxns)

    Contents uL
    10 uM VF2 primer 1.25 uL
    10 uM VR primer 1.25 uL
    DNA 0.5 uL
    Q5 MM 12.5 uL
    Water 19.5 uL

8/9

Gel ran

Lanes:

  1. Ladder
  2. R0011
  3. B001
  4. K1033
  5. R008
  6. J61100
  7. C0012
  8. K592
  • Didn't work

Nanodrop of Miniprepped Plamids:

    Tube # Concentration (ng/ul)
    1 277.3
    2 #
    2 #
    2 #
PCR of Minprepped Plasmids
    Contents ul
    Q5 Buffer 5uL
    H2O 16.25 uL
    10 uM F Primer 1.25 uL
    10 uM R Primer 1.25 uL
    Construct 0.5 uL
    Q5 Polymerase 0.25 uL
    Q5 Polymerase 0.25 uL

PCR Settings:

    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30s
    Denaturation 98 20s
    Annealing 56 15s
    Extension 72 30s
    Final Extension 72 60s

8/10

25 ul PCR of Miniprepped Plasmids for Project

    Contents ul
    Q5 Buffer 5
    Q5 Polymerase 5
    H2O 16.25
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Construct 0.5
    Phase Temperature (°C) Time (sec)
    Initial Denaturation 98 30
    Denaturation 98 20
    Annealing 56 15
    Extension 72 30
    Final Extension 72 60
PCR was run on the following construct concentrations in ng/ul
  • 273
  • 176.5
  • 296.6
  • 311.5
  • 301.6
  • 301.6
  • 259
  • 366.6
  • PCRs did not work → Changed annealing temperature to 66°C
  • Re-ran the first 5 constructs on the above list

25 ul PCR of Miniprepped Plasmid for BioBrick Portion

    Contents ul
    Q5 Master Mix 12.5
    H2O 9.5
    10 uM F Primer 1.25
    10 uM R Primer 1.25
    Template 0.5
  • This setup was used for all BioBrick Parts
  • Ran with the "Sunil" Program with a 66°C annealing temperature

    8/11

    8/12

    Today was a depressing day

    8/13

    8/14