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EPF-Lausanne/7 October 2009
From 2009.igem.org
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==Wet Lab== | ==Wet Lab== | ||
| + | ===Colony PCR=== | ||
| + | On the same double transformants -> 2 min extension. | ||
| + | ===Gel=== | ||
| + | Here the 2nd strains seems to have a double transformant. But we let the gel overnight so there is no BrEth any more, we had to take a time of exposure of 38.2 s to see something. We will do a nicer gel this afternoon. | ||
| + | |||
| + | ===Preparation of the time-course experiment=== | ||
Prepared cells for time-course experiment: | Prepared cells for time-course experiment: | ||
| Line 45: | Line 51: | ||
The cells were put in the incubator at 37°C in their respective experimental conditions. Measurements of OD and fluorescence will be taken every 30 min starting at 1h of incubation, so that we have a time-course measurement. For the last sample, we will do a kinetic measurement overnight to see the decay of the RFP fluroescence. | The cells were put in the incubator at 37°C in their respective experimental conditions. Measurements of OD and fluorescence will be taken every 30 min starting at 1h of incubation, so that we have a time-course measurement. For the last sample, we will do a kinetic measurement overnight to see the decay of the RFP fluroescence. | ||
| + | ===Miniprep=== | ||
| + | We did a miniprep of the Trp-mutated strains that should be double transformants to extract the DNA and make a digestion assay in order to confirm the gel's results : | ||
| + | - RO1.1 + BB1 JRG 1046 (n°1,3,7) | ||
| - | + | - RO2.4 + BB1 JRG 1046 (n°1,3,6) | |
| - | + | Also miniprepped DH5 alpha RO2.4+BB1 clone n.3 to extract the 2 working plasmids (LovTap and read-out 2), so that we can use this DNA directly for future transformations. | |
| - | + | ||
| - | + | ===Digestion assay=== | |
| + | For each clones, we use digestion by P and S (since LovTAP contains 2 P sites and Trp op contains 2 S sites). For reaction, incubate at 37°C (INCUB37). Digestion of about 1h30. | ||
| + | ===Gel=== | ||
| + | Once again to see the double transformants + of digestion assay. | ||
| + | Result ?? TO BE COMPLETED | ||
| + | |||
| + | |||
| + | |||
| + | ===Results of the plate-reader experiment=== | ||
| + | Graphic : | ||
| + | |||
| + | [[Image:071009_dh5alpha_ro2_dt.jpg|center|thumb|upright=4]] | ||
==People in the lab== | ==People in the lab== | ||
Revision as of 07:36, 8 October 2009
Contents |
Wet Lab
Colony PCR
On the same double transformants -> 2 min extension.
Gel
Here the 2nd strains seems to have a double transformant. But we let the gel overnight so there is no BrEth any more, we had to take a time of exposure of 38.2 s to see something. We will do a nicer gel this afternoon.
Preparation of the time-course experiment
Prepared cells for time-course experiment:
- 7mL of fresh LB + 1mL of overnight cell culture (DH5 alpha RO2.4 + BB1 clone n.3)
- 5 different conditions:
- +light +IPTG -Trp
- +light -IPTG -Trp
- -light +IPTG -Trp
- -light -IPTG +Trp
- -light -IPTG -Trp
The cells were put in the incubator at 37°C in their respective experimental conditions. Measurements of OD and fluorescence will be taken every 30 min starting at 1h of incubation, so that we have a time-course measurement. For the last sample, we will do a kinetic measurement overnight to see the decay of the RFP fluroescence.
Miniprep
We did a miniprep of the Trp-mutated strains that should be double transformants to extract the DNA and make a digestion assay in order to confirm the gel's results :
- RO1.1 + BB1 JRG 1046 (n°1,3,7)
- RO2.4 + BB1 JRG 1046 (n°1,3,6)
Also miniprepped DH5 alpha RO2.4+BB1 clone n.3 to extract the 2 working plasmids (LovTap and read-out 2), so that we can use this DNA directly for future transformations.
Digestion assay
For each clones, we use digestion by P and S (since LovTAP contains 2 P sites and Trp op contains 2 S sites). For reaction, incubate at 37°C (INCUB37). Digestion of about 1h30.
Gel
Once again to see the double transformants + of digestion assay.
Result ?? TO BE COMPLETED
Results of the plate-reader experiment
Graphic :
People in the lab
Gab, Tu
