Team:Groningen/Labwork/24 July 2013



Run the samples of the PCR made 23-07 over a 1.5% agarose gel. The samples show the presence of the expected bands for 3 out of 4 signal sequences. So it is tried to obtain these samples by Gel extraction. No bands are seen on gel. Made a restriction digestion of the rest of the samples of silk 2 and 3 (strepF-R and F-strepR) to obtain a higher concentration. With the restriction digest a ligation to the four signal sequences is made. The primers that are ordered for heat motility are found and diluted. A PCR is made to obtain Pdes, CheY RBS, the samples are present. So a PCR purification is done. New PCR's are made for all three silks and the four signal sequences.


Made a -80 stock of the Munich BBa_k823023 backbone + our IPTG promoter.
Made a miniprep of the inoculated cells with the k077557 biobrick that Chicago requested.
Concentration of the final sample is 21.1 ng/uL.
Sample was stored in -20 freezer

Determined concentration of PCR samples pdes and cheY by nanodrop. pdes 151.2 ng/uL and cheY 71.4 ng/uL.
Ran a 1.5% gel with both samples, at 90 V for 24 minutes. well 1:marker, well 2:cheY, well 3:pdes.


The plates are taken out from the incubator and the presence of RFP is detected using UV light.