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Team:IPN-UNAM-Mexico/Notebook/July
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==='''''08-July-2009'''''=== | ==='''''08-July-2009'''''=== | ||
| - | * | + | *We made the restrictions and put them on an agarose gel with 10 wells for 40 min. |
| - | *3μl DNA | + | *We used 3μl DNA plus 2μl Buffer. |
| - | *We | + | *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 | + | *Run on only Plasmidic DNA but we have no DNA. |
---- | ---- | ||
Revision as of 06:58, 21 October 2009
Contents |
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 for the digestion we did a 17 hours incubation at 37°C.
- 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 made the restrictions and put them on an agarose gel with 10 wells for 40 min.
- We 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
- Starting again, and take off DNA from the folder plating R0079, C0178, C0179, C0060, B0034, I739001 biobriks,
- Left 16 hours in 37ºC.
10-July-2009
We don’t have transformed cells in this point we have to delay and request 2009 catalog.
28-July-2009
With the 2009 catalog we retake the work and take on the biobriks we need. First of all we test them to be sure that the biobriks worked properly, for this we take a biobrik with RFP reporter (BBa_I3522), plate and incubate 17 hours in 37ºC.
29-July-2009
The transformation was successful we are sure the biobricks and method works and we proceed to take off our biobricks as next chart.
| 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 use the same method as follow.
- Elusion: 3μl DNA from the well.
- Put it on a Eppendorf Vial 1.5 ml with competent cells.
- Electoporate shock to transform and recovery in LB ampr 1 hour and a half.
- Plate on petri dishes and incubate 17 hours.
31-July-2009
- Plated Top 10 biobricks F1610, K091146, K081016, K081009, R0051, K081018 and incubate during 17 hours
