Team:TU Delft/12 August 2010 content
From 2010.igem.org
Contents |
Lab work
Alkane degradation
BioBricks number 014K and 020A (the latter was already tried before but failed) are up today. We will do a digestion and a ligation followed by a transformation in order to make these BioBricks. Next to these two BioBricks we will also be working on placing some of the previously made BioBricks in the 1C3 plasmid so we can ship it to the registry.
Digestion
# | Sample | Enzyme 1 | Enzyme 2 | Enzyme 3 | Buffer | BSA | Needed fragment |
1 | 011A | EcoRI | SpeI | AseI | 2 (Biolabs) | ✓ | ‘E - J61100-alkB2-J61100-rubA3 - S’ |
2 | 013K | EcoRI | XbaI | ✗ | 3 (Biolabs) | ✓ | ‘E - J61100-rubA4-J61100-rubR-B0015-pSB1AK3 - X’ |
3 | 017A | EcoRI | SpeI | AseI | 2 (Biolabs) | ✓ | ‘E - J61100-ladA - S’ |
4 | 018A | EcoRI | XbaI | ✗ | 3 (Biolabs) | ✓ | ‘E - J61101-ADH-pSB1A2 - X’ |
5 | 011A | EcoRI | PstI | AseI | 3 (Biolabs) | ✗ | ‘E - J61100-alkB2-J61100-rubA3 - P’ |
6 | 013K | EcoRI | PstI | HindIII | 2 (Biolabs) | ✗ | ‘E - J61100-rubA4-J61100-rubR-B0015 - P’ |
7 | 021 | EcoRI | PstI | SmaI | 3 (Biolabs) | ✓ | ‘E - J61107-ALDH-B0015 - P’ |
8 | 402 | EcoRI | PstI | AseI | 3 (Biolabs) | ✗ | ‘E - J61101-PhPFDα - P’ |
9 | 403 | EcoRI | PstI | AseI | 3 (Biolabs) | ✗ | ‘E - J61101-PhPFDβ - P’ |
10 | pSB1C3 | EcoRI | PstI | ✗ | 3 (Biolabs) | ✗ | ‘E - pSB1C3 - P’ |
The digestions were done during 2 hours, and checked on a gel:
[[Image:|300px|thumb|left|Digestion check]]
Ligation
Directly following the digestion, the products were ligated for 4 hours:
# | BioBrick | Fragment 1 | Fragment 2 / Plasmid |
1 | 014K | ‘E - J61100-alkB2-J61100-rubA3 - S’ | ‘E - J61100-rubA4-J61100-rubR-B0015-pSB1AK3 - X’ |
2 | 020A | ‘E - J61100-ladA - S’ | ‘E - J61101-ADH-pSB1A2 - X’ |
3 | 011C | ‘E - J61100-alkB2-J61100-rubA3 - P’ | ‘E - pSB1C3 - P’ |
4 | 013C | ‘E - J61100-rubA4-J61100-rubR-B0015 - P’ | ‘E - pSB1C3 - P’ |
5 | 021C | ‘E - J61107-ALDH-B0015 - P’ | ‘E - pSB1C3 - P’ |
5 | 402C | ‘E - J61101-PhPFDα - P’ | ‘E - pSB1C3 - P’ |
5 | 403C | ‘E - J61101-PhPFDβ - P’ | ‘E - pSB1C3 - P’ |
10 μL of this ligation mix were used to transform Top10 competent cells.