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Team:TU Delft/3 August 2010 content
From 2010.igem.org
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The digestions were done during 2 hours, and checked on a gel: | The digestions were done during 2 hours, and checked on a gel: | ||
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| + | [[Image:TUDelft_20100803_digestion.png|300px|thumb|left|Digestion check]] | ||
Lane description | Lane description | ||
Revision as of 14:28, 3 August 2010
Alkane degradation
Yesterday's digestions showed that BioBrick 007 was not the expected length. From this it was concluded that the plasmid thus does not contain the desired insert, so we will make the BioBrick again. This was done, starting with a digestion. An extra cut was done in the backbone of alkB2 to decrease the chances of religation.
| # | Sample | Enzyme 1 | Enzyme 2 | Enzyme 3 | Buffer | BSA | Needed fragment |
| 1 | J61100 | SpeI | PstI | ✗ | 2 (Biolabs) | ✓ | ‘S – pSB1A2 - J61100– P’ |
| 2 | 1 μg alkB2 | XbaI | PstI | AseI | 2 (Biolabs) | ✓ | ‘X – alkB2 – P’ |
The digestions were done during 2 hours, and checked on a gel:
Lane description
| # | Description | Expected Length (bp) | Status |
| 0 | Smartladder (5μl) | ||
| 1 | ‘S – pSB1A2 - J61100 – P’ | 2116 | ✓ |
| 2 | undigested pSB1A2-J61100 | ✓ | |
| 3 | ‘X – alkB2 – P’ | 1263 | ✓ |
| 4 | Undigested alkB2 | ✓ |
Directly following the digestion, the products were ligated for 4 hours:
| # | BioBrick | Fragment 1 | Fragment 2 |
| 1 | pSB1A2-J61100-alkB2 | ‘X – alkB2 – P’ | ‘S – pSB1A2-J61100 – P’ |
10 μL of the ligation mix was used to transform Top10 competent cells.
