Team:TU Delft/15 July 2010 content
From 2010.igem.org
Line 5: | Line 5: | ||
===Emulsifier=== | ===Emulsifier=== | ||
- | The results from the Colony PCR were not conclusive [https://2010.igem.org/Team:TU_Delft#/blog?blog=14_July_2010 yesterday]. That is why we decided to isolate the plasmid using [[Team:TU_Delft/protocols/birnboim_plasmid_isolation|Birnboim protocol]]. | + | The results from the Colony PCR were not conclusive [https://2010.igem.org/Team:TU_Delft#/blog?blog=14_July_2010 yesterday]. That is why we decided to isolate the plasmid using [[Team:TU_Delft/protocols/birnboim_plasmid_isolation|Birnboim protocol]]. We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]]. |
The following plasmid concentrations were obtained: | The following plasmid concentrations were obtained: |
Revision as of 19:54, 19 July 2010
Lab work
Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing
Some of transformations of yesterday containing the different ligations gave colonies (1 a 2 per plate). We picked as many colonies as possible of every plate and performed a colony PCR overnight to check which colonies contained the right insert. At the same time we grow them overnight in 5 mL LB medium containing the appropriate antibiotic.
Emulsifier
The results from the Colony PCR were not conclusive yesterday. That is why we decided to isolate the plasmid using Birnboim protocol. We used 3 mL of the bacterial cells to make -80 °C stocks.
The following plasmid concentrations were obtained:
BioBrick | Composed of | Concentration (ng/μL) |
K398203 | OprG-B0015 (transformant 6) | |
K398203 | OprG-B0015 (transformant 7) | |
K398204 | AlnA-B0015 (transformant 11) |
The plasmids were subsequently digested with an enzyme that characteristic digest for all different plasmids. The plasmids were digested with HindIII. This should yield 3 bands in the lane with the plasmid pSB1T3 containing J04450 (neg control), 1 band in the lane with AlnA-B0015 and 2 bands in the lane with OprG-B0015 on gel.
# | Digestion reaction | Used Buffer |
1 | 1.5 μg pSB1T3 | Buffer 2 + BSA (BioLabs) |
2 | 1 μg OprG-B0015 (transformant 6) | Buffer 2 + BSA (BioLabs) |
3 | 1 μg OprG-B0015 (transformant 7) | Buffer 2 + BSA (BioLabs) |
4 | 1 μg AlnA-B0015 (transformant 11) | Buffer 2 + BSA (BioLabs) |
1% Agarose gel of digestion products:
Lane description:
# | Description | Expected Length (bp) |
1 | SmarLadder marker (5 μL) | |
2 | Undigested pSB1T3 (5 μL + 1 μL loadingbuffer) | |
3 | Digested pSB1T3 (10 μL + 1 μL loadingbuffer) | 1972, 1176 |
4 | Undigested OprG-B0015 (transformant 6) (5 μL + 1 μL loadingbuffer) | |
5 | Digested OprG-B0015 (transformant 6) (10 μL + 2 μL loadingbuffer) | 1828, 1475, 222 |
6 | Digested OprG-B0015 (transformant 7) (10 μL + 2 μL loadingbuffer) | 1828, 1475, 222 |
7 | Undigested AlnA-B0015 (transformant 11) (5 μL + 1 μL loadingbuffer) | |
8 | Digested AlnA-B0015 (transformant 11) (10 μL + 2 μL loadingbuffer) | 3511 |