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Team:TU Delft/29 July 2010 content
From 2010.igem.org
Contents |
Lab work
Hydrocarbon Sensing
Cell bank
A cell bank was made from E.coli Top10 cells transformed with AlkS-E0422 and AlkS-B0015. We used 2 mL of the bacterial cells to make -80 °C stocks.
Alkane Degradation
PCR Amplification
K398007A, K398008A, K398009A, K398010A, K398017A, K398018A, K398019A, B0015 were amplified with the universal primers G00100 and G00101. The product was put on 1% agarose gel:
Lane Description:
| # | Description | Expected Length (bp) | Primers | Status |
| M1 | SmartLadder | n/a | n/a | n/a |
| 1 | PCR product of 007 | 1616 | G00101 + G00101 | ✓ |
| 2 | PCR product of 008 | 473 | G00101 + G00101 | ✓ |
| 3 | PCR product of 009 | 482 | G00101 + G00101 | ✓ |
| 4 | PCR product of 010 | 1505 | G00101 + G00101 | ✓ |
| 5 | PCR product of 017 | 1625 | G00101 + G00101 | ✓ |
| 6 | PCR product of 018 | 1052 | G00101 + G00101 | ✓ |
| 7 | PCR product of 019 | 1796 | G00101 + G00101 | ✓ |
| 8 | PCR product of B0015 | 445 | G00101 + G00101 | ✓ |
Digestion
The PCR products were subsequently digested:
| # | Sample | Enzyme 1 | Enzyme 2 | Buffer | BSA | Needed fragment |
| 1 | 40 μL PCR product 007 | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–007–S’ |
| 2 | 40 μL PCR product 008 | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–008-P’ |
| 3 | 40 μL PCR product 009 | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–009–S’ |
| 4 | 40 μL PCR product 010 | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–010-P’ |
| 5 | 40 μL PCR product 017 | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–009–S’ |
| 6 | 40 μL PCR product 018 | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–018-P’ |
| 7 | 40 μL PCR product 019 | EcoRI | SpeI | 2 (BioLabs) | ✓ | ‘E–019-S’ |
| 8 | 40 μL PCR product B0015 | XbaI | PstI | 2 (BioLabs) | ✓ | ‘X–B0015-P’ |
Ligation
The digestion products were ligated overnight:
| # | BioBrick | Fragment 1 | Fragment 2 | Final volume |
| 1 | K398011 | 4 μL ‘E–007–S’ | 4 μL ‘X–008–P’ | 10 μL |
| 2 | K398012 | 4 μL ‘E–009–S’ | 4 μL ‘X–010–P’ | 10 μL |
| 3 | K398020 | 4 μL ‘E–017–S’ | 4 μL ‘X–018–P’ | 10 μL |
| 4 | K398021 | 4 μL ‘E–019–S’ | 4 μL ‘X–B0015–P’ | 10 μL |
| 5 | negative control | 4 μL ‘E–007–S’ | 4 μL H2O | 10 μL |
Plasmid isolation
A plasmid isolation was done on the positive colonies of yesterday. This was done using a Qiagen Miniprep kit. We used 2 mL of the bacterial cells to make -80 °C stocks.
