Team:Heidelberg LSL/Notebook-10/03/2012

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Notebook

Welcome to our notebook!

Here you will find the documentation of our laboratory work of the last few month in diary form. This notebook comprises the work in three phases:

Biosensor Construction - 10/03/2012

• Information: Standard cloning - our Strategy
  • GFP and LacZ were digested with EcoRI (E) and Xbal (X)
  • RecA was digested with EcoRI and SpeI (S)
  • SulA was annealed, so it has the overhangs for EcoRI (as well as the whole BBa prefix) and a XbaI compatible overhang

(PLACEHOLDER: schema of cloning)

• Gelelectrophoresis of the digested biobrick parts in order to extract the GFP and LacZ backbone as well as recA
  

Protocol: In the meantime, the restriction digests were mixed with 6 µl of 6 x Loading Dye (fermentas). The whole restriction digest was loaded onto a 1 % agarose gel. Therefor, the agarose gel was prepared by adding 2 g of agarose to 200 ml of 1x TBE buffer and heated up in a microwave for 2 min/600 W. Thereby, the agarose was melted. Afterwards, the gel was stirred on a magnetic stirring device until the solution reached ~ 60°C and then the gel was poured. 10 µl of ethidium bromide were subsequently added by our supervisor Katharina Genreith (as EtBr is very toxic, we preferred our supervisor to do the handling of that when running a gel for the first time). The samples were then loaded on the gel together with 1kb plus loading ladder (fermentas) and run for 1 h @ 100V. The expected bands can be seen on the figure (picture constructed using the WinSerial Cloner software).

(PLACEHOLDER: figure showing the gel we expect)

(PLACEHOLDER: photo of the gel)

• Gel extraction of the indicated band (backbones for LacZ and GFP as well as recA insert) was done using a Qiagen gel extraction kit.
  

Protocol: The bands were cut of the gel under UV light (caution: wear goggles in order to protect your eyes!) using a scalpel and the extracted band was put into a 2 ml eppi cup. Afterwards, 600 µl of buffer QG were added to each gel band and the mixture was incubated on a thermomixer device at 50 °C and 700 rpm shaking for 20 min. Afterwards, 200 µl of Isopropanol were added to the mix containing recA (as recA is pretty short compared to the others, isopropanol is required). Afterwards, the whole reaction mix was loaded onto a gel extraction column and centrifuged for 1 min/max speed; flow-through was discarded. The column was subsequently washed with 500 µl of buffer QG and then 750 µl buffer PE, always centrifuging the column at max speed for 1 min at each washing step and discarding the flow-through. Finally, DNA was eluted in 20 µl of water.

• Concentration measurement of the pre-cut parts using Nanodrop