Glasgow/Wetlab/Week6
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Week 6
Monday 6th August 2007
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Maia checked the presence and size of (*m*) and (*s*) inserts within the Topo vector by repeating the PCR performed on 02/08. Results confirmed presence of inserts with the correct size. The TOPO vectors containing (*m*) and (*s*) were then sent for sequencing to ensure the inserts did not contain any mistakes. DNA sequenced and the primers used are summarized in the table below.
Label B1 B3 C5 Gene (*m*) (*s*) (*m*) Colony 17,18,19,26 20 25,24 Primers (*m*)_for_1 + Methyl _2 (*s*)_for_1 + Oxy_2 (*m*)_for_1 + (*m*)_rev_1 (NB - For each 2 x 10µl was sent)
- Christine repeated the cloning of the 7-gene operon into TOPO vector using fresh PCR product. The PCR performed was a repeat of the PCR from 31/07. 4 µl of PCR product was cloned into TOPO vector. (see Protocol 12)
Tuesday 7th August 2007
- Christine transformed TOP10 cells with TOPO vector containing the 7 gene operon.
- (*d→g*) were amplified again with KOD XL and KOD using the respective programs (see Protocol 9). Primers used: (*d*)_for_1 and (*g*)_rev_1 .Expected size is 3.5 kb.
Wednesday 8th August 2007
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A digest scheme was designed to allow for the insertion of our biobricks into construction vectors.
Biobricks : DntR, (*s*), (*m*), XylR, XylR + Pr, Pr, Pu Construction Vectors: 3/20G, 4/6B, 4/50
Thursday 9th August 2007
- Each biobrick and construction vector was digested with EcoRI and SpeI (Roche)(see Protocol 7)
- Digest products were resolved on 1% agarose gels and either gel extracted (see Protocol 11) or PCR purified (see Protocol 14)
- Each biobrick was ligated with three different construction vectors (listed above) using the Quick Ligation Kit (New England Biolabs) (see Protocol 15)
- Ligation products were transformed into TOP10 cells and grown on LB agar plates supplemented with appropriate antibiotic
Friday 10th August 2007
Results of construction vector transformations: (number of colonies per plate)
| Construction Vector | |||
|---|---|---|---|
| Biobrick | 3/20G | 4/50 | 4/6B |
| Pu | 0 | 2 | 3 |
| Pr | 0 | V.small | V.small |
| XylR | 2 | 3 | V.small |
| XylR + Pr | 0 | 0 | 0 |
| DntR | 5 | 3 | 3 |
| (*m*) | 12 | V.small | 8 |
| (*s*) | 31 | 1 | 56 |
