Team:UCL/Labbook/Week17

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<div class="full_page">
<div class="full_page">
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<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week19"> Week 19</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week20"> Week 20</a>
+
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a>  
</p>  
</p>  
</div>
</div>
 +
 +
<p class="minor_title">Week 17</p>
 +
<div class="full_row">
 +
<div class="gap">
 +
</div>
 +
<p class="body_text">
 +
<b>Bacterial Labs</b>
 +
</p>
 +
<p class="body_text">
 +
<b>Monday 23rd September</b>
 +
</p>
 +
<p class="body_text">
 +
Nanodrop readings
 +
</p>
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Sample</th>
 +
<th>ng/ul</th>
 +
<th>260/280</th>
 +
</tr>
 +
<tr>
 +
<td>MMP9</td>
 +
<td>382.5</td>
 +
<td>1.86</td>
 +
</tr>
 +
<tr>
 +
<td>ZEC</td>
 +
<td>401</td>
 +
<td>1.49</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<div class="gap">
 +
</div>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Nanodrops of the 6 colonies</th>
 +
<th>ng/ul</th>
 +
<th>260/280</th>
 +
</tr>
 +
<tr>
 +
<td>1x1</td>
 +
<td>22.3</td>
 +
<td>1.46</td>
 +
</tr>
 +
<tr>
 +
<td>1x2</td>
 +
<td>78.5</td>
 +
<td>1.45</td>
 +
</tr>
 +
<tr>
 +
<td>1x3</td>
 +
<td>11.0</td>
 +
<td>1.77</td>
 +
</tr>
 +
<tr>
 +
<td>1x4</td>
 +
<td>33.9</td>
 +
<td>1.50</td>
 +
</tr>
 +
<tr>
 +
<td>1x5</td>
 +
<td>35.2</td>
 +
<td>1.55</td>
 +
</tr>
 +
<tr>
 +
<td>5x1</td>
 +
<td>28.7</td>
 +
<td>1.53</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<p class="body_text">
 +
<b>Tuesday 24th September</b>
 +
</p>
 +
<p class="body_text">
 +
CMV PCR with Taq polymerase
 +
</p>
 +
<p class="body_text">
 +
-10 reactions and 1 control
 +
</p>
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Reaction components</th>
 +
<th>Tubes 1-10</th>
 +
<th>Control (11)</th>
 +
</tr>
 +
<tr>
 +
<td>Taq Buffer</td>
 +
<td>5</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>dNTP</td>
 +
<td>1</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>F Primer</td>
 +
<td>1</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>R Primer</td>
 +
<td>1</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Template (pSecTag2A, 1ng/ul)</td>
 +
<td>1.5</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Taq polymerase</td>
 +
<td>0.25</td>
 +
<td>0.25</td>
 +
</tr>
 +
<tr>
 +
<td>dH2O</td>
 +
<td>40.25</td>
 +
<td>41.75</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<p class="body_text">
 +
Tubes were labelled from 1 to 11.
 +
This was unsuccessful.
 +
</p>
 +
 +
<p class="body_text">
 +
Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9.
 +
</p>
 +
 +
<p class="body_text">
 +
<b>Sunday 29th September</b>
 +
</p>
 +
<p class="body_text">
 +
There were not enough colonies for B3&4. The rest of the samples were <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> minipreped</a>
 +
and digested and run on a gel cut.
 +
</p>
 +
<p class="body_text">
 +
4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Nanodrop</a> readings of these inoculations were poor.
 +
</p>
 +
 +
</div>
 +
 +
<div class="gap">
 +
</div>
 +
 +
<div class="full_row">
 +
<div class="gap">
 +
</div>
 +
<p class="body_text">
 +
<b>Mammalian Labs</b>
 +
</p>
 +
<p class="body_text">
 +
<b>Monday 23rd September</b>
 +
</p>
 +
<p class="body_text">
 +
Seeded 2x105 cells (1x105 cells/ml) per well into 16 wells set in three 6-well plates. Stock Hela passaged (P21).
 +
</p>
 +
 +
 +
<p class="body_text">
 +
24th September
 +
</p>
 +
<p class="body_text">
 +
CMV PCR with Taq polymerase
 +
</p>
 +
<p class="body_text">
 +
-10 reactions and 1 control
 +
 +
</p>
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Reaction Components</th>
 +
<th>Tubes 1-10</th>
 +
<th>Control 11</th>
 +
</tr>
 +
<tr>
 +
<td>Taq buffer</td>
 +
<td>5</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td>dNTP</td>
 +
<td>1</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>F primer</td>
 +
<td>1</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>R primer</td>
 +
<td>1</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>Template (pSecTag2A, 1 ng/ul)</td>
 +
<td>1.5</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>Taq polymerase</td>
 +
<td>0.25</td>
 +
<td>0.25</td>
 +
</tr>
 +
<tr>
 +
<td>Water</td>
 +
<td>40.25</td>
 +
<td>41.25</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>50</td>
 +
<td>50</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<p class="body_text">
 +
This was unsuccessful.
 +
</p>
 +
 +
<p class="body_text">
 +
<b>25th September</b>
 +
</p>
 +
<p class="body_text">
 +
Aim: To stably transfect Hela cells with ’’Str Ble’’.
 +
</p>
 +
<p class="body_text">
 +
Confluency of dishes: 30-50%
 +
 +
</p>
 +
<p class="body_text">
 +
Per well of the 6-well plates: 13 well total.
 +
</p>
 +
<p class="body_text">
 +
1.    Mass of DNA: 2.0µg
 +
</p>
 +
<p class="body_text">
 +
2.    Volume of DNA dissolved in TE Buffer: 5.0 µl
 +
</p>
 +
<p class="body_text">
 +
3.    Final volume of DNA diluted in serum free media: 100 µl
 +
</p>
 +
<p class="body_text">
 +
4.    Volume of superfect (SF) reagent:10.0 µl
 +
</p>
 +
<p class="body_text">
 +
5.    Volume of serum-containing media: 600 µl
 +
</p>
 +
<p class="body_text">
 +
2x ”mock transfected” wells (No DNA, No SE)
 +
</p>
 +
<p class="body_text">
 +
Procedure:
 +
</p>
 +
<p class="body_text">
 +
Made a ”mask mix” solution for the 13 well-plates to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transfected</a>, with the following composition :
 +
</p>
 +
<p class="body_text">
 +
1.    34 ng
 +
</p>
 +
<p class="body_text">
 +
2.    93.5 ul
 +
</p>
 +
<p class="body_text">
 +
3.    1870 ul
 +
</p>
 +
<p class="body_text">
 +
4.    187 ul
 +
</p>
 +
<p class="body_text">
 +
5.    11220 ul
 +
 +
</p>
 +
<p class="body_text">
 +
Transfer DNA-Buffer solution to 15 ml falcon tube. Add non-serum media (1776.5µl), Filter, Add SF, Vortex (10.6), Incubate for 5-10 min at room temperature. Meanwhile aspirate and wash cells with 4ml PBS. Add media with serum (600ml). Pipette up and down x2. Apply to cells(≈700µl). Incubate for 2-3 hours. Change media. Incubate (24 hours)
 +
</p>
 +
<p class="body_text">
 +
Note : for characterisation , add 1µl of 100µg/ml zeocin to one plate, 2µl to another, and 3µl to the last to make up the concentrations to those over the page.
 +
</p>
 +
<p class="body_text">
 +
<b>26th September</b>
 +
</p>
 +
<p class="body_text">
 +
Characterisation:
 +
</p>
 +
<p class="body_text">
 +
24 hours after exposure:
 +
</p>
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Viability (%)</th>
 +
<th>50 µg/ml Zec</th>
 +
<th>100 µg/ml Zec</th>
 +
<th>150 µg/ml Zec</th>
 +
</tr>
 +
<tr>
 +
<td>Control</td>
 +
<td>80</td>
 +
<td>80</td>
 +
<td>70</td>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>80</td>
 +
<td>80</td>
 +
<td>80</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>75</td>
 +
<td>80</td>
 +
<td>75</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>75</td>
 +
<td>80</td>
 +
<td>75</td>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>70</td>
 +
<td>80</td>
 +
<td>75</td>
 +
</tr>
 +
</table>
 +
</p>
 +
 +
<p class="body_text">
 +
<b>Sunday 29th September</b>
 +
</p>
 +
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>24 Hours</th>
 +
<th>A (%)</th>
 +
<th>F (%)</th>
 +
<th>48 Hours</th>
 +
<th>A (%)</th>
 +
<th>F (%)</th>
 +
<th>72 Hours</th>
 +
<th>A (%)</th>
 +
<th>F (%)</th>
 +
</tr>
 +
<tr>
 +
<td>C1</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>C1</td>
 +
<td>0</td>
 +
<td>4</td>
 +
<td>C1</td>
 +
<td>0</td>
 +
<td>10</td>
 +
</tr>
 +
<tr>
 +
<td>C2</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>C1</td>
 +
<td>0</td>
 +
<td>5</td>
 +
<td>C1</td>
 +
<td>5</td>
 +
<td>20</td>
 +
</tr>
 +
<tr>
 +
<td>C3</td>
 +
<td>0</td>
 +
<td>2</td>
 +
<td>C3</td>
 +
<td>0</td>
 +
<td>4</td>
 +
<td>C3</td>
 +
<td>1</td>
 +
<td>40</td>
 +
</tr>
 +
<tr>
 +
<td>P1</td>
 +
<td>0</td>
 +
<td>3</td>
 +
<td>P1</td>
 +
<td>0</td>
 +
<td>5</td>
 +
<td>P1</td>
 +
<td>5</td>
 +
<td>50</td>
 +
</tr>
 +
<tr>
 +
<td>P2</td>
 +
<td>0</td>
 +
<td>2</td>
 +
<td>P2</td>
 +
<td>0</td>
 +
<td>10</td>
 +
<td>P2</td>
 +
<td>1</td>
 +
<td>15</td>
 +
</tr>
 +
<tr>
 +
<td>P3</td>
 +
<td>0</td>
 +
<td>1</td>
 +
<td>P3</td>
 +
<td>0</td>
 +
<td>10</td>
 +
<td>P3</td>
 +
<td>1</td>
 +
<td>50</td>
 +
</tr>
 +
</table>
 +
 +
</div>
 +
 +
<!-- END CONTENT ------------------------------------------------------------------------------------------------------>
<!-- END CONTENT ------------------------------------------------------------------------------------------------------>

Latest revision as of 03:52, 5 October 2013

Lab Weeks

Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18

Week 17

Bacterial Labs

Monday 23rd September

Nanodrop readings

Sample ng/ul 260/280
MMP9 382.5 1.86
ZEC 401 1.49

Nanodrops of the 6 colonies ng/ul 260/280
1x1 22.3 1.46
1x2 78.5 1.45
1x3 11.0 1.77
1x4 33.9 1.50
1x5 35.2 1.55
5x1 28.7 1.53

Tuesday 24th September

CMV PCR with Taq polymerase

-10 reactions and 1 control

Reaction components Tubes 1-10 Control (11)
Taq Buffer 5 5
dNTP 1 1
F Primer 1 1
R Primer 1 1
Template (pSecTag2A, 1ng/ul) 1.5 0
Taq polymerase 0.25 0.25
dH2O 40.25 41.75

Tubes were labelled from 1 to 11. This was unsuccessful.

Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9.

Sunday 29th September

There were not enough colonies for B3&4. The rest of the samples were minipreped and digested and run on a gel cut.

4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. Nanodrop readings of these inoculations were poor.

Mammalian Labs

Monday 23rd September

Seeded 2x105 cells (1x105 cells/ml) per well into 16 wells set in three 6-well plates. Stock Hela passaged (P21).

24th September

CMV PCR with Taq polymerase

-10 reactions and 1 control

Reaction Components Tubes 1-10 Control 11
Taq buffer 5 5
dNTP 1 1
F primer 1 1
R primer 1 1
Template (pSecTag2A, 1 ng/ul) 1.5 0
Taq polymerase 0.25 0.25
Water 40.25 41.25
Total 50 50

This was unsuccessful.

25th September

Aim: To stably transfect Hela cells with ’’Str Ble’’.

Confluency of dishes: 30-50%

Per well of the 6-well plates: 13 well total.

1. Mass of DNA: 2.0µg

2. Volume of DNA dissolved in TE Buffer: 5.0 µl

3. Final volume of DNA diluted in serum free media: 100 µl

4. Volume of superfect (SF) reagent:10.0 µl

5. Volume of serum-containing media: 600 µl

2x ”mock transfected” wells (No DNA, No SE)

Procedure:

Made a ”mask mix” solution for the 13 well-plates to be transfected, with the following composition :

1. 34 ng

2. 93.5 ul

3. 1870 ul

4. 187 ul

5. 11220 ul

Transfer DNA-Buffer solution to 15 ml falcon tube. Add non-serum media (1776.5µl), Filter, Add SF, Vortex (10.6), Incubate for 5-10 min at room temperature. Meanwhile aspirate and wash cells with 4ml PBS. Add media with serum (600ml). Pipette up and down x2. Apply to cells(≈700µl). Incubate for 2-3 hours. Change media. Incubate (24 hours)

Note : for characterisation , add 1µl of 100µg/ml zeocin to one plate, 2µl to another, and 3µl to the last to make up the concentrations to those over the page.

26th September

Characterisation:

24 hours after exposure:

Viability (%) 50 µg/ml Zec 100 µg/ml Zec 150 µg/ml Zec
Control 80 80 70
1 80 80 80
2 75 80 75
3 75 80 75
4 70 80 75

Sunday 29th September

24 Hours A (%) F (%) 48 Hours A (%) F (%) 72 Hours A (%) F (%)
C1 0 1 C1 0 4 C1 0 10
C2 0 1 C1 0 5 C1 5 20
C3 0 2 C3 0 4 C3 1 40
P1 0 3 P1 0 5 P1 5 50
P2 0 2 P2 0 10 P2 1 15
P3 0 1 P3 0 10 P3 1 50

Retrieved from "http://2013.igem.org/Team:UCL/Labbook/Week17"