Team:TU Delft/15 July 2010 content

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===Emulsifier===
===Emulsifier===
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The results from the Colony PCR were not conclusive [http://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]].
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The results from the Colony PCR were not conclusive [http://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:

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