Team:UCL/Labbook/Week17

From 2013.igem.org

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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>
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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>
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Revision as of 13:47, 4 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.

Wednesday 25th September

Thursday 26th September

Friday 27th September

Saturday 28th September

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

Mammalian Lab

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 tubeà 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 -

Mammalian Lab

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

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