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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 9
Bacterial Lab
29th July - A glycerol stock of pSecTag2A from 2009 was restored. This was streaked onto 3 plates (2x LB Amp and 1x No drug), additionally four Falcons with 2ul LB were inoculated with the glycerol stock (2x LB No drug, 2X LB Amp). These were left to incubate overnight.
30th July - pSecTag2A Amp and No drug cultures were centrifuged (5 minutes at 4000rpm) and the pellet frozen to be used later in a miniprep. pSecTag2A plates displayed good colony growth. Colonies were picked from non-competent W3110 streaked plates and inoculated in LB ->incu-shake 37C o/n.
| Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
|---|---|---|---|
| 1 (Main) | Yes | Yes | 100+ |
| 2 (Positive Control) | No | Yes | 100+ |
| 3 (Negative Control) | Yes | No | 0 |
31st July - A new stock of competent cells were generated and stored, these were tested for competence via transformation using pSecTag2A and streaking onto amp plates -> incubate 37C o/n.
Ampicillin was produced and stored. Following miniprep of pSecTag2A a gel was prepared for analytical digest with HindIII [link to HindIII conditions] [insert gel image HindIII analytical digest of pSecTag2A]. 50X TAE diluted to 1X.
| 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
29th July - Thawed and revived HeLa cells, grown in two T25 flasks in DMEM + 10% FBS + 2 mM L-Glu
30th July - HeLa cells are not looking very happy, with many floating cells. Cells are left to grow over the weekend
August
Bacterial Lab
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 left for incubation 37C o/n.
Transformation of competent cells with pSecTag2A. 100µl was then spread onto 6 plates (4x Amp, 2x No drug). Left to incubate o/n 37C.
2nd August - Plates displayed significant colony growth on amp plates indicating successful transformation. Negative control however displayed slight colony growth - ampicillin not working effectively, new ampicillin was therefore prepared. End of week inventory was recorded and stocks were topped up.
| Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
|---|---|---|---|
| 1 (Main) | Yes | Yes | 100+ |
| 2 (Positive Control) | No | Yes | 100+ |
| 3 (Negative Control) | Yes | No | 15 |
