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Team:UCL/Labbook/Week17
From 2013.igem.org
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Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9. | Made new inoculations from 5X CMP and 1X CMP plates for Ligated CMV-MMP9. | ||
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| - | There were not enough colonies for B3&4. The rest of the samples were minipreped and digested and run on a gel cut. | + | 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. | ||
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| - | 4 more inoculations in 10 ml LB broth of A2, A5, B2 and C2. Nanodrop readings of these inoculations were poor. | + | 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. |
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| - | + | <b>25th September</b> | |
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| - | Made a ”mask mix” solution for the 13 well-plates to be transfected, with the following composition : | + | 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 : |
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| - | Transfer DNA-Buffer solution to 15 ml falcon | + | 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) |
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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. | 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. | ||
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| - | <b> | + | <b>26th September</b> |
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24 hours after exposure: | 24 hours after exposure: | ||
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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 |
