From 2010.igem.org

Lab work

Ordered DNA

The transformations of 19 July containing the different ligations gave colonies (~5 per plate). We picked 5 colonies per plate and performed a colony PCR.

Alkane degradation

Unfortunately there were no transformants on yesterday's plates. This is most likely due to the fact that the ligation didn't work with the small pieces of DNA of the RBSs. Next plan is to cut open the RBS plasmid without removing the RBS (with SpeI and PstI) and insert the gene in this plasmid. We will try this tomorrow.

Salt Tolerance

The overnight ligation was transformed using the standard method and grown up overnight at 37 degrees C.

Emulsifier

2% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 μL + 2 μL loadingbuffer was loaded. 5 μL was loaded of marker

Lane Description

Unfortunately all other lanes were empty or the same as the negative control. So from this gel we conclude that the ligation has failed.

Characterisation of Anderson RBS sequences

Assembly of reference construct

Method 1

Hypothesis II The transformants that were replated on kanamycin plates produced colonies, indicating that hypothesis II was invalid. To test whether these colonies did contain the proper insert however, a colony PCR was performed:

1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 5 µL was loaded with 1 µL loadingbuffer. 5 µL was loaded of marker

Lane description:

Method 3

TOP10 competent cells were transformed with yesterday's ligation product.

Method 4

The obtained colonies on ampicillin plates were screened for GFP fluorescence. No discernable fluorescence was seen under UV excitation. It was decided to perform a single colony PCR on 17 randomly selected colonies to check for the proper insert:

1% agarose of colony PCR. Gel run at 100V for 1 hour. Of all samples 5 µL was loaded with 1 µL loadingbuffer. 5 µL was loaded of marker

Lane description:

It appeared that the 17 colonies sampled did not contain the expected insert.

Solvent Tolerance and Hydrocarbon Sensing

Characteristic digestion of isolated plasmids

To determine whether the plasmids are correct. Characteristic cuts were made using the restriction enzyme PvuI.

Results of the digestion on 1% agarose gel

1% agarose of plasmid check using digestion reaction. Gel runned at 100V for 1 hour. Of all samples 10 μL was loaded with 2 μL loadingbuffer. 5 μL was loaded of marker

Lane description:

Unfortunately most of the lanes showed incorrect bands. So from this gel we conclude that the ligation has failed.