Team:IPN-UNAM-Mexico/Notebook/July
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- | The transformation was successful we are sure the biobricks and | + | 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: |
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Revision as of 07:14, 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, we extracted DNA from the folder plates R0079, C0178, C0179, C0060, B0034, I739001.
- Left for 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 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