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Team:TU Delft/6 July 2010 content
From 2010.igem.org
Contents |
Lab work
Ordered DNA stocks
The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!
We were so excited; we immediately dissolved the DNA in water and performed a transformation with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.
- AlkB2 (Ampicillin)
- rubA3 (Ampicillin)
- rubA4 (Ampicillin)
- rubB (Kanamycin)
- ladA (Ampicillin)
- ADH (Ampicillin)
- ALDH (Kanamycin)
- bbc1 (Ampicillin)
- AlnA (Ampicillin)
- OprG (Ampicillin)
- PalkS1-2 (Ampicillin)
- PalkB (Ampicillin)
- P(CaiF) (Ampenicillin)
- AlkS (Kanamycin)
- PhPFD-α (Ampicillin)
- PhPFD-β (Ampicillin)
Characterization of Anderson RBS sequences
Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were digested:
| # | Digestion reaction | Used Buffer | Needed fragment |
| C1 | 1.15 μg J61100 + SpeI + PstI | Buffer 2 (BioLabs) | ‘S – J61100 - pSB1A2 – P’ |
| C2 | 1.68 μg J61101 + SpeI + PstI | Buffer 2 (BioLabs) | ‘S – J61101 - pSB1A2 – P’ |
| C3 | 1.30 μg J61107 + SpeI + PstI | Buffer 2 (BioLabs) | ‘S – J61107 - pSB1A2 – P’ |
| C4 | 0.95 μg J61117 + SpeI + PstI | Buffer 2 (BioLabs) | ‘S – J61117 - pSB1A2 – P’ |
| C5 | 0.51 μg J61127 + SpeI + PstI | Buffer 2 (BioLabs) | ‘S – J61127 - pSB1A2 – P’ |
| D1 | 2.59 μg I13401 + XbaI + PstI | Buffer 2 (BioLabs) | ‘X – I13401 – P’ |
| D2 | 2.65 μg I13401 + XbaI + PstI | Buffer 2 (BioLabs) | ‘X – I13401 – P’ |
The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a 1% agarose gel for comparison with the non-digested BioBricks:
Lane description:
| # | Description | Expected Length (bp) | Remarks |
| 1 | SmartLadder marker (5 μL) | n/a | |
| 2 | Undigested J61100 (1 μL + 4 μL MQ + 1 μL Loading Buffer) | Circular Plasmid | |
| 3 | Undigested J61101 (1 μL + 4 μL MQ + 1 μL Loading Buffer) | Circular Plasmid | |
| 4 | ‘S – J61100 - pSB1A2 – P’ | 2116 | Visible |
| 5 | ‘S – J61101 - pSB1A2 – P’ | 2116 | Visible |
| 6 | Undigested J61107 (1 μL + 4 μL MQ + 1 μL Loading Buffer) | Circular plasmid | |
| 7 | ‘S – J61107 - pSB1A2 – P’ | 2116 | Visible |
| 8 | Undigested J61117 (1 μL + 4 μL MQ + 1 μL Loading Buffer) | Circular plasmid | |
| 9 | ‘S – J61117 - pSB1A2 – P’ | 2116 | Visible |
| 10 | Undigested J61127 (1 μL + 4 μL MQ + 1 μL Loading Buffer) | Circular plasmid | |
| 11 | ‘S – J61127 - pSB1A2 – P’ | 2116 | Visible |
| 12 | Undigested I13401 (1 μL + 4 μL MQ + 1 μL Loading Buffer) | Circular plasmid | |
| 13 | ‘X – I13401 – P’ | 883 | Visible |
| 14 | empty | empty | empty |
| 15 | BioRad EZ Load | n/a | Visible |
Followed by over night ligation:
| # | BioBrick | Recipient plasmid | Fragment insert | Final volume |
| 1 | K398500A | 130 μg ‘S – J61100 - pSB1A2 – P’ | 154 μg ‘X-I13401-P’ | 26 μL |
| 2 | K398501A | 206 μg ‘S – J61101 - pSB1A2 – P’ | 247 μg ‘X-I13401-P’ | 25 μL |
| 3 | K398502A | 196 μg ‘S – J61107 - pSB1A2 – P’ | 247 μg ‘X-I13401-P’ | 25 μL |
| 4 | K398503A | 201 μg ‘S – J61117 - pSB1A2 – P’ | 247 μg ‘X-I13401-P’ | 25 μL |
| 5 | K398504A | 202 μg ‘S – J61127 - pSB1A2 – P’ | 247 μg ‘X-I13401-P’ | 25.5 μL |
| 6 | Ligase Control (J61117 - pSB1A2) | 123 μg ‘S – J61117 - pSB1A2 – P’ | None | 15 μL |
To all samples one-tenth of the total volume ligase buffer was added.
Emulsifier
Pieter is testing different condition for the emulsifying Assay. To determine the emulsifying activity we set up a calibration curve with SDS. He prepared the following samples:
| # | Hexane (mL) | Tris Buffer pH 8 (mL) | 10% SDS (mL) |
| B | 1 | 1.1 | 0 |
| 100 | 1 | 1 | 0.1 |
| 50 | 1 | 1.05 | 0.05 |
| 25 | 1 | 1.075 | 0.025 |
| 10 | 1 | 1.09 | 0.01 |
| 5 | 1 | 1.095 | 0.005 |
Kampioenen!!!!
Today the Dutch team will play the semi-final of the World Cup! Hear us cheer! Stop sound
