Revision as of 02:29, 27 October 2007

Wet Lab: Protocols: Refined Testing for AHL Detection

Aims

To determine the response of the pTet-LuxR-pLux-GFP construct, in-vitro, for different concentrations of AHL
To determine the response time of the construct in-vivo, for different AHL concentrations

First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
Place the 96 well plates together with their plate mates in their respective incubators so as to heat them up to the appropriate temperature before the experiments start.
For the next step of the go to the biochemistry level 5 and remove:
- A.A's from kits
- Premix tubes (140ul)
- S30 tubes (150ul)
Prepare the following cell extract mixture for all 21 reactions(one extra reaction):
- First add 420µl of the premix solution into a labelled eppendorf.
- Then add 315µl of the S30 cell extract mixture to S30 Premix Without Amino Acid
- Then prepare a complete amino acid mixture: Add 52.5μl of two amino acid minus mixtures into the eppendorf with the premix and S30 solution. Each amino acid minus mixture is missing one type of amino acid, and so by combining two solutions we are complementing each solution for the missing amino acid. Place eppendorf in a rack on bench.
- Vortex the tubes to mix thoroughly
- Any left over premix or cell extract should be returned to the freezer in biochemistry level 5 and labeled with new volumes.
Prepare the following dilution to get DNA concentration of 4µg:
- Put 174µl of pLux DNA into a labelled eppendorf
- Add 166µl of nuclease free water into the DNA
- This will give a total volume of 340&micr0;l, which is enough for all 18 samples with DNA in them

First read the background fluorescence of the 96-well plate using the fluorometer.
Choose suitable wells, with minimum fluorescence (30-40 au) to put the samples in. Don't use the wells at the edges and avoid putting samples in consecutive wells.
Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
Remove lid off the 96 well plate and place in the fluorometer. Create a file name insert temp under: D:\IGEM\INSERT DATE\ID\ 25oC. Export the data here. Each file should be named as the following:
- construct-temp-time-date
This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
Then to begin the reaction add 4ug of purified DNA sample to each well indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
Add the appropriate amount of AHl to each well, so that the total reaction volume in each well is 60ul.
Place the plate in the fluorometer to measure its initial fluorescent reading.
After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
Repeat the measurement every 30 minutes, for 6 hours.

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