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EPF-Lausanne/8 September 2009
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==Wet Lab== | ==Wet Lab== | ||
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===Cultures=== | ===Cultures=== | ||
| + | |||
'''Results:''' | '''Results:''' | ||
:RO1 has grown in LB and M9 | :RO1 has grown in LB and M9 | ||
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:RO2+BB #1,4 did not grow | :RO2+BB #1,4 did not grow | ||
:RO2+BB #3 has grown in LB but not in M9 | :RO2+BB #3 has grown in LB but not in M9 | ||
| - | -> problem with our glycerol stock. We did some plates of the cultures that grew. | + | -> problem with our glycerol stock (RO2 + BB didn't grow in LB !!). We did some plates of the cultures that grew. |
| + | |||
| + | For safety we did some plates with the glycerol stocks of RO2 + BB 1,4,8. We also did some plates with the cultures that grew (RO1 1,2,3, RO2 4,5,10, RO2 + BB 3). | ||
| + | |||
<br> | <br> | ||
===Characterization=== | ===Characterization=== | ||
| - | As RO1 grew in both media, we put it in 25mL | + | As RO1 grew in both media, we put it in 25mL medium with Amp. We did also with RO2 (LB), and RO2+BB (LB), and LacI-RFP. |
| + | |||
| + | For the characterisation, we did different combinations : -TRP, -TRP -ATC, +TRP -ATC, +TRP +ATC. Every time for all the clones in M9 and then in LB. | ||
| + | |||
| + | TRP : LB : dilution 50x so 1ul, M9 : dilution 25x so 2 ul. We took the stock solution 4,250 g/L. | ||
| + | |||
| + | ATC : 5x dilution so 10 ul from the 500 ng/ml stock solution. | ||
| + | |||
| + | The aim was to get used to the machine and to see the differences between LB or M9. | ||
| + | |||
| + | Here are the results of the characterization : | ||
| + | |||
| + | RO1#1 + TRP in LB | ||
| + | There is already some TRP in LB so it shouldn't change a lot. | ||
| + | [[Image:RO11TRPLB.jpg|center|RO1#1 + TRP in LB]] | ||
| + | |||
| + | <br> | ||
| + | RO1#1 without TRP in LB | ||
| + | This doesn't mean a lot as there is already some TRP in LB but we can try to see a difference with RO1#1 + TRP in LB. | ||
| + | [[Image:RO11LB.jpg|center|RO1#1 in LB]] | ||
| + | |||
| + | <br> | ||
| + | RO1#2 + TRP in LB | ||
| + | [[Image:RO12TRPLB.jpg|center|RO1#2 + TRP in LB]] | ||
| + | |||
| + | <br> | ||
| + | RO1#2 without TRP in LB | ||
| + | [[Image:RO12LB.jpg|center|RO1#2 in LB]] | ||
| + | |||
| + | <br> | ||
| + | RO1#3 + TRP in LB | ||
| + | The clone 3 doesn't seem to be functional. | ||
| + | [[Image:RO13TRPLB.jpg|center|RO1#3 + TRP in LB]] | ||
| + | |||
| + | <br> | ||
| + | RO1#3 without TRP in LB | ||
| + | [[Image:RO13LB.jpg|center|RO1#3 in LB]] | ||
| + | |||
| + | <br> | ||
| + | RO1#3 + TRP in M9 | ||
| + | [[Image:RO13TRPM9.jpg|center|RO1#3 + TRP in M9]] | ||
| + | |||
| + | <br> | ||
| + | RO1#3 without TRP in M9 | ||
| + | [[Image:RO13M9.jpg|center|RO1#3 in M9]] | ||
| + | |||
| + | <br> | ||
| + | RO2 + TRP + ATC | ||
| + | [[Image:RO2TRPATC.jpg|center|RO2 + TRP + ATC]] | ||
| + | |||
| + | <br> | ||
| + | RO2 + TRP - ATC | ||
| + | [[Image:RO2TRP.jpg|center|RO2 + TRP]] | ||
| + | |||
| + | <br> | ||
| + | RO2 without TRP + ATC | ||
| + | [[Image:RO2ATC.jpg|center|RO2 + ATC]] | ||
| + | |||
| + | <br> | ||
| + | RO2 without TRP and without ATC | ||
| + | [[Image:RO2without.jpg|center|RO2]] | ||
| + | |||
| + | ===Results of the transformation=== | ||
| + | They all grew : RO1#2 + BB5, RO1#1 + BB1, RO1#3 + BB3. | ||
| + | |||
| + | So we did a colony PCR to be sure that we have the double transformation, using the Taq platinium protocole. | ||
| + | |||
| + | We picked 8 clones of RO1#2 + BB5, 8 clones of RO1#1 + BB1 and 5 clones of RO1#3 + BB3. | ||
| + | |||
| + | We ran a gel to check if it has the two plasmids : for the clones that worked, we should see two bands. We ran a gel : we should get two fragments. We can see that all our clones worked (had the two constructs), except one : clone 5 for RO1#3 + BB3. | ||
| + | |||
| + | ===Transformation=== | ||
| + | Then we did a transformation of the ligation products : BB1, BB3, BB5, BB6 in a chlorophenical receptor, on Chl plates. | ||
| + | |||
| + | ===Culturing=== | ||
| + | LB : RO2 + BB (1,3,4,8), LacI-RFP (#1,2), BB/Chl (results of the gel : 4 clones). | ||
| + | M9 : LacI-RFP (2 clones), RBS BBa_B0030 (control for the fluo. of the cells), RO2 (4,5,10), RO1 (1,2,3). | ||
==People in the lab== | ==People in the lab== | ||
Latest revision as of 16:51, 20 October 2009
Contents |
Wet Lab
Cultures
Results:
- RO1 has grown in LB and M9
- RO2 has grown in LB but not in M9
- RO2+BB #1,4 did not grow
- RO2+BB #3 has grown in LB but not in M9
-> problem with our glycerol stock (RO2 + BB didn't grow in LB !!). We did some plates of the cultures that grew.
For safety we did some plates with the glycerol stocks of RO2 + BB 1,4,8. We also did some plates with the cultures that grew (RO1 1,2,3, RO2 4,5,10, RO2 + BB 3).
Characterization
As RO1 grew in both media, we put it in 25mL medium with Amp. We did also with RO2 (LB), and RO2+BB (LB), and LacI-RFP.
For the characterisation, we did different combinations : -TRP, -TRP -ATC, +TRP -ATC, +TRP +ATC. Every time for all the clones in M9 and then in LB.
TRP : LB : dilution 50x so 1ul, M9 : dilution 25x so 2 ul. We took the stock solution 4,250 g/L.
ATC : 5x dilution so 10 ul from the 500 ng/ml stock solution.
The aim was to get used to the machine and to see the differences between LB or M9.
Here are the results of the characterization :
RO1#1 + TRP in LB There is already some TRP in LB so it shouldn't change a lot.
RO1#1 without TRP in LB
This doesn't mean a lot as there is already some TRP in LB but we can try to see a difference with RO1#1 + TRP in LB.
RO1#2 + TRP in LB
RO1#2 without TRP in LB
RO1#3 + TRP in LB
The clone 3 doesn't seem to be functional.
RO1#3 without TRP in LB
RO1#3 + TRP in M9
RO1#3 without TRP in M9
RO2 + TRP + ATC
RO2 + TRP - ATC
RO2 without TRP + ATC
RO2 without TRP and without ATC
Results of the transformation
They all grew : RO1#2 + BB5, RO1#1 + BB1, RO1#3 + BB3.
So we did a colony PCR to be sure that we have the double transformation, using the Taq platinium protocole.
We picked 8 clones of RO1#2 + BB5, 8 clones of RO1#1 + BB1 and 5 clones of RO1#3 + BB3.
We ran a gel to check if it has the two plasmids : for the clones that worked, we should see two bands. We ran a gel : we should get two fragments. We can see that all our clones worked (had the two constructs), except one : clone 5 for RO1#3 + BB3.
Transformation
Then we did a transformation of the ligation products : BB1, BB3, BB5, BB6 in a chlorophenical receptor, on Chl plates.
Culturing
LB : RO2 + BB (1,3,4,8), LacI-RFP (#1,2), BB/Chl (results of the gel : 4 clones). M9 : LacI-RFP (2 clones), RBS BBa_B0030 (control for the fluo. of the cells), RO2 (4,5,10), RO1 (1,2,3).
People in the lab
Mélanie, Caroline, Basile, Nicolas
