User:Maira Tariq/sandbox

Protocols for DNA concentration experiments

Experiments to be carried out are to determine the optimum concentration of the pLux-luxR-pLux-GFP construct for the ID, in-vitro.

The concentrations of DNA that will be tested are: 1, 2, 4, 6 and 9µg. Each concentration of DNA will be tested over a period of 6 hours, as it is expected that the system will respond within about 2-3 hours to AHL. The evaporation of the samples will be taken into account when analysing the data.

Aims

• To determine the concentration of DNA for which the response to AHL being induced is optimum, in terms of the reponse time and the output fluorescence at the response time.

Equipment

• Fluorometer + PC
• 25°C water bath
• Fluorometer plate
• Gilson pipettes 200, 20, 10
• Eppendorf Tubes x 7
• Stopwatch
• Foil
• Clear tape

Reagents

• S30 E.coli Cell Extract
• Nuclease Free water
• DNA

Preparation of reactions

1. First collect all equipment and reagents and ensure that the fluorometer and the PC connected has a data collection protocol installed.
2. Place the 96well plate into the 25°C water bath
3. For the cell extract, get the following out of the cell extract kit:
   • A.A's from kits
   • Premix tube
   • S30 tubes
4. To prepare the commercial E.coli Cell Extract, carry out the following Procedure:
   
   1. First prepare a complete amino acid mixture for the extract solution: Add the 30µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 60µl. Each amino acid minus mixture is missing one type of amino acid.
   2. Take an eppendorf tube and add the 60µl of the E.coli complete amino acid mixture.
   3. Add 240µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
   4. Then add 180µl of S30 Extract Circular too.
   5. This mixture is for all the samples. Label the tube.
   6. Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
5. Incubate the prepared cell extract mixtures in the water bath set at 25°C.
6. Prepare a 36µl of 50nM solution of AHL for all the DNA concentrations:
   1. Aliquot 1.8µl of 1mM AHL into an eppendorf tube.
   2. Add in 34.2µl of water into the eppendorf to get the required dilution and label it.
7. Add the diluted AHL into the eppendorf tube with the cell extract and place back in the water bath.
8. Prepare the different DNA concentrations (concentration of pLux DNA = 460ng/µl):
   1. Concentration 1 = 1µg: Add 2.17µl of DNA in 14.83µl nuclease free water.
   2. Concentration 2 = 2µg: Add 4.35µl of DNA in 12.65µl nuclease free water.
   3. Concentration 3 = 4µg: Add 8.70µl of DNA in 8.30µl nuclease free water.
   4. Concentration 4 = 6µg: Add 13.04µl of DNA in 3.96µl nuclease free water.
   5. Concentration 5 = 7.5µg: Add 16.3µl of DNA in 0.7µl nuclease free water.
9. Put 34µl of each DNA concentration into a seperate, labeled eppendorf tube and place them in the 25°C water bath.

Loading Plate

1. Take the plate out of the incubation.
2. Follow the schematic for the plate and begin by loading 43µl of the in vitro expression system with AHL into each of the wells.
3. Tap down the top of the plate to bring down any solution to bottom of the well.
4. Then add 17µl of purified DNA sample to each well, as indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
5. Add 17µl of distilled water into the two negative conrtol wells, as shown in the schematics.
6. Put 60µl of water into some empty wells in the middle of the plate. These will be used to check for evaporation.
7. After the DNA and the cell extract mixtures have been put into their respective wells, load the program on the PC to measure the fluorescence in the right wells.
8. Create a file with name referring to the temperature of the plate, under: D:\IGEM\INSERT DATE\ID\ OTR. The data from the fluoreometer will be exported here.
9. Each file with the reading should be named as the following:
   • construct-temp-time-date
10. While the program loads, get the plate out of the water bath and wipe off the water on it.
11. Take a reading in the fluorometer. Before each measurement remember to tap down the solution and to remove the clear tape on it before placing in the fluorometer.
12. As soon as the reading has been taken, unload the plate and place the clear tape on the plate and place back in the water bath. Cover the plate with foil to prevent the DNA from getting bleached due to light. Make sure that the plate is not outside the water bath for longer than 5mins. Remember to close the plate holder of the fluorometer after each reading.
13. After 30 mins of incubation, load the program on the PC again, to measure the fluorescence in the right wells.
14. Take another fluorescence reading, repeating steps 9-13.
15. Take a reading similarly every half an hour, until 6 hours have elapsed since the first reading.
16. After the last reading, measure the amount of water left in the wells (with no cell extract mixture) to check the amount of fluid that has evaporated.
17. Wash off the plates with 70% ethanol and rinse with distilled water

Schematic

Well	Test Construct	Concentration of DNA	In vitro chassis
	Distilled Water + AHL (Negative control)	0µg	Commercial E.coli extract
	Distilled Water + AHL (Negative control)	0µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	1µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	1µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	2mg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	2µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	4µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	4µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	6µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	6µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	9µg	Commercial E.coli extract
	pLux-luxR-pLux-GFP + AHL	9µg	Commercial E.coli extract

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