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EPF-Lausanne/5 August 2009
From 2009.igem.org
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From the transformations of yesterday, only 3/16 plates worked: the "classic method" LovTap-Term, its negative control (probably the Term plasmid religated), and one clone on the LacI-RBS-LovTap-Term!!! We suspect that the death cassette plasmid doesn't work correctly (since this hasn't been successful from the beginning), so next time we'll use other backbones for the ligation. | From the transformations of yesterday, only 3/16 plates worked: the "classic method" LovTap-Term, its negative control (probably the Term plasmid religated), and one clone on the LacI-RBS-LovTap-Term!!! We suspect that the death cassette plasmid doesn't work correctly (since this hasn't been successful from the beginning), so next time we'll use other backbones for the ligation. | ||
| - | We did a colony PCR (with the iGEM primers) on 20 clones from the LovTap-Term, 5 from the negative control, and the one from the LacI-RBS-LovTap-Term, and ran the resulting fragments on an agarose gel to see whether the inserts were the expected ones. | + | We did a colony PCR (with the iGEM primers) on 20 clones from the LovTap-Term, 5 from the negative control, and the one from the LacI-RBS-LovTap-Term, and ran the resulting fragments on an agarose gel to see whether the inserts were the expected ones. Result: only one of the 20 clones (LacI-RBS) was good. |
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| + | We did a liquid culture of the right clone. | ||
To redo the ligations for what didn't work, we did the "1,5-step PCR" again and the normal PCR with the 3 LacI-RBS we have. | To redo the ligations for what didn't work, we did the "1,5-step PCR" again and the normal PCR with the 3 LacI-RBS we have. | ||
Revision as of 09:34, 10 August 2009
Contents |
Wet Lab
From the transformations of yesterday, only 3/16 plates worked: the "classic method" LovTap-Term, its negative control (probably the Term plasmid religated), and one clone on the LacI-RBS-LovTap-Term!!! We suspect that the death cassette plasmid doesn't work correctly (since this hasn't been successful from the beginning), so next time we'll use other backbones for the ligation.
We did a colony PCR (with the iGEM primers) on 20 clones from the LovTap-Term, 5 from the negative control, and the one from the LacI-RBS-LovTap-Term, and ran the resulting fragments on an agarose gel to see whether the inserts were the expected ones. Result: only one of the 20 clones (LacI-RBS) was good.
We did a liquid culture of the right clone.
To redo the ligations for what didn't work, we did the "1,5-step PCR" again and the normal PCR with the 3 LacI-RBS we have.
People in the lab
Nath, Basile, Gab, Christian, Nicolas
