Glasgow/Wetlab/Week3
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Week 3
Tuesday 17th July 2007
- Restriction Digests of transformed BioBricks (see Protocol 7):
| Label | BioBrick | Plasmid | Description | Enzymes | Expected Sizes |
|---|---|---|---|---|---|
| 3/19A | BBa_J23119 | pSB1A2 | strong constitutive promoter | NheI, PvuI | 680bp, 1430bp |
| 1/9G | BBa_R0062 | pBB1A2 | HSL and luxR | EcoRI, PvuI | 1460bp, 660bp |
| 1/16P | BBa_J04500 | pSB1AK3 | IPTG inducer and RBS | PvuII, PvuI | 2250bp, 1030bp, 730bp |
| 4/11C | BBa_p1010 | pSB3K3 | death gene | BamHI, XhoI | 190bp, 2390bp,840bp |
| 1/5H | BBa_E0040 | pSB1A2 | GFP no promoter | Hime II, PvuI | 1630bp, 1170bp |
| 4/6D | BBa_I52001 | p5B4K5 | High copy death gene | AvaI, PvuI | 920bp, 1470bp, 2120bp |
Wednesday 18th July 2007
- Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
- Maija ran a gel of the restriction digests to check the sizes of the biobricks. These were the maps we made yesterday.
- Maia is extracting DNA from 3 samples of Pseudomonas Aeruginosa, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see Protocol 8); only change was to shorten "shaking" time from 10 to 2 minutes.
- PCR trial run using Reddymix and Touch 2 done (see Protocol 9 for PCR).
| Template | Primers |
|---|---|
| pGLTUR |
|
| pQF52 | DntR_prefix + DntR_suffix |
| DmpR WT/24 | DntR_prefix + DntR_suffix |
- Biobricks: [http://partsregistry.org/Part:BBa_R0062 R0062], [http://partsregistry.org/Part:BBa_E0040 E0040], [http://partsregistry.org/Part:BBa_J04500 J04500], [http://partsregistry.org/Part:BBa_J23119 J23119], [http://partsregistry.org/Part:BBa_p1010 p1010], [http://partsregistry.org/Part:BBa_I52001 I52001].
Thursday 19th July 2007
- Maia redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (see Protocol 9) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
- XylR prefix and XylR suffix
- Pr prefix and Pr suffix
- Pr prefix and XylR suffix
- Pu prefix and Pu suffix
- DntR prefix and DntR suffix 2
Friday 20th July 2007
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