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Team:UCL/Labbook/Week9
From 2013.igem.org
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 9
Bacterial Lab
Monday 29th July - A glycerol stock of pSecTag2A from 2009 was restored. This was streaked onto 3 plates (2x LB Amp and 1x No drug). Four Falcons with 2ul LB were inoculated overnight at 37°C with the glycerol stock (2x LB No drug, 2X LB Amp).
Tuesday 30th July
pSecTag2A Ampcillin and No drug cultures were centrifuged (5 minutes at 4000rpm) and the pellet frozen to be used later in a miniprep. pSecTag2A plates displayed colony growth. Colonies were picked from non-competent W3110 streaked plates and inoculated in LB.
| Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
|---|---|---|---|
| 1 (Main) | Yes | Yes | 100+ |
| 2 (Positive Control) | No | Yes | 100+ |
| 3 (Negative Control) | Yes | No | 0 |
Wednesday 31st July
A new stock of competent cells were generated and stored, these were tested for competence via transformation using pSecTag2A and streaking onto ampicillin plates. Incubate at 37°C overnight.
Ampicillin was produced and stored. Following miniprep of pSecTag2A a gel was prepared analytical digest with HindIII.
| Item | Volume (ul) |
|---|---|
| DNA pSecTag2A | 5 |
| HindIII | 1 |
| Buffer | 1 |
| BSA | 0.5 |
| dH20 | 2.5 |
| Total | 10 |
Results displayed uncut bands at expected lengths. However HindIII cut pSecTag2A wells displayed too many bands, indicating possible contamination or uncut DNA.
Mammalian Lab
Monday 29th July
Thawed and revived HeLa cells, grown in two T25 flasks in DMEM + 10% FBS + 2 mM L-Glu
Tuesday 30th July
HeLa cells are not looking very happy, with many floating cells. Cells are left to grow over the weekend
August
Bacterial Lab
Thursday 1st August
Results from newly generated competent cells transformed with pSecTag2A:
| Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
|---|---|---|---|
| 1 (Main) | Yes | Yes | 100+ |
| 2 (Positive Control) | No | Yes | 100+ |
| 3 (Negative Control) | Yes | No | 10 |
This indicated that the cells are more competent than the last batch, although growth on the negative control was a cause for concern. Therefore the transformation protocol from yesterday was repeated to see whether the ampicilin was working. Plates were left for incubation at 37°C overnight.
Transformation of competent cells with pSecTag2A. 100µl was then spread onto 6 plates (4x Amp, 2x No drug). Left to incubate overnight at 37°C.
Friday 2nd August
Plates displayed significant colony growth on ampicillin plates indicating successful transformation. Negative control however displayed slight colony growth, indicating that the ampicillin was not working effectively. New ampicillin was therefore prepared.
| Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
|---|---|---|---|
| 1 (Main) | Yes | Yes | 100+ |
| 2 (Positive Control) | No | Yes | 100+ |
| 3 (Negative Control) | Yes | No | 15 |
