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Team:UC Davis/Notebook/Week 8
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Revision as of 19:19, 25 August 2011
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--Monday 8/01/11--
Our transformations yielded colonies, but there were much more than expected on our vector control plates. We decided to PCR screen 4 colonies from each of the the ligations. After running them on a gel, it looks as though some of these colonies were successful ligations! At the end of the day, we cultured E0240+I13453 so that we could proceed with more construction tomorrow.
--Tuesday 8/02/11--
After miniprepping the cultured E0240+I13453, we set up a fast digest such that we could try a triple ligation. Our goal parts were: E0240+I13453+C0040, E0240+I13453+C0012, and E0240+I13453+C0051. We cut both E0240+I13453 and our repressor sequences as inserts and ligated them into a pSB1C3 backbone. We transformed and plated the ligation products.
--Wednesday 8/03/11--
Our triple ligation plates don't look very good. Most plates only have one colony while our vector control (pSB1C3) has about three. Today, our plan is to attempt to construct the same parts but by using our usual approach with fast digest enzymes. We digested, ligated, transformed, and plated these parts. Hopefully tomorrow we will see better results.
--Thursday 8/04/11--
Once again, our plated transformation were not ideal. The vector controls revealed that there was quite a bit of background on our plates, so we decided to PCR screen colonies once again. We were able to get a few good colonies from this screen, but overall it was not very successful. This lead us to try digesting, ligating, and transforming once more. We will probably stop attempting to use fast digest enzymes, as over the past few weeks they have given us unsuccessful results. Additionally, while digesting with normal restriction enzymes, we will try to allow them to incubate for much longer than three and a half hours (up to 7).
--Friday 8/05/11--
In the morning we set up a normal digestion and will purify, ligate, and transform it on Sunday.
