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Team:IPN-UNAM-Mexico/Notebook/July
From 2009.igem.org
July
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07-July-2009
- Digestion of the following biobricks were done with EcoRI (E), SpeI (S), and XbaI (X).
| Biobrick | Enzyme restriction |
|---|---|
| R0079 | E, S |
| F1610 | E, X |
| B0034 | E, S |
| C0078 | E, X |
| C0079 | E, X |
| B0015 | E, X |
- We propose this restrictions for standard assembly and digestion we did a 17 hours incubation in 37°C. camera
- Digest Mix:
| EcoRI - SpeI | Per reaction |
|---|---|
| Plasmidic DNA | 3μl |
| Enzyme EcoRI | 2μl |
| Enzyme SpeI | 2μl |
| Buffer NBE | 2μl |
| BSA | 0.5μl |
| H2O | 10.5μl |
| Total | 20μl |
| EcoRI - XbaI | Per reaction |
|---|---|
| Plasmidic DNA | 3μl |
| Enzyme EcoRI | 2μl |
| Enzyme XbaI | 2μl |
| Buffer NBE | 2μl |
| BSA | 0.5μl |
| H20 | 10.5μl |
| Total | 20μl |
08-July-2009
- We did the restrictions and put them on an agarose gel with 10 wells for 40 min.
- Used 3μl DNA plus 2μl Buffer.
- We didn't get any DNA from the gel so maybe the DNA volume is too low. We tried again with 20μl DNA plus 3μl Buffer
- Run on only Plasmidic DNA but we have no DNA.
09-July-2009
- Started again, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001.
- Left 16 hours at 37°C.
10-July-2009
We didn't have any transformed cells so at this point we had to delay wetlab work and request 2009 catalog.
28-July-2009
After the 2009 catalog arrived we could resume the wetlab work because some of the biobricks we needed were not available on previous folders. First of all we tested them to be sure that the biobricks worked properly, for this we took a biobrick with RFP reporter (BBa_I3522), plated and incubated for 17 hours at 37ºC.
29-July-2009
The transformation was successful, so we are now sure that the biobricks and methods we used work correctly so the next step is to extract the biobricks we are going to use for our project which are the following:
| Number | Biobrick |
|---|---|
| 2 | BBa_R0079 |
| 3 | BBa_F1610 |
| 4 | BBa_K091146 |
| 5 | BBa_K093005 |
| 6 | BBa_K081016 |
| 7 | BBa_EC840 |
| 8 | BBa_K081009 |
| 9 | BBa_R0051 |
| 10 | BBa_J06800 |
| 11 | BBa_Q04121 |
| 12 | BBa_B0034 |
| 13 | BBa_C0079 |
| 14 | BBa_C0179 |
| 15 | BBa_B0015 |
| 16 | BBa_K081018 |
| 17 | BBa_K116640 |
For transformation we used the following method:
- Elution: 3μl DNA from the well.
- Put it on a 1.5 ml Eppendorf vial with competent cells.
- Transformation was made by electroporation and recovery in LB media with Ampr for 1 hour and a half.
- Plate on petri dishes and incubate 17 hours.
31-July-2009
- E. coli Top10 cells which were transformed with biobricks F1610, K091146, K081016, K081009, R0051 and K081018 were plated and incubated for 17 hours
