From 2007.igem.org

Protocols for DNA concentration experiments

Experiments to be carried out are to determine the optimum concentration of the CBD construct, in-vitro, so that we get the highest level of protein expression after a period of 6hours. The construct to be tested is pTet-GFP.

The concentrations of DNA that will be tested are: 1, 2, 4 and 6µg.

Aims

• To determine the concentration of pTet construct for which output is optimum, in terms of the reponse time and the output fluorescence at the end of the experiment time.

Equipment

• Fluorometer + PC
• 37°C incubator
• Fluorometer plate (black)
• Sticky seal tape
• Gilson pipettes 200, 20, 10
• Eppendorf Tubes x 7
• Stopwatch
• Foil

Reagents

• Commercial S30 E.coli extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• Nuclease Free water x1ml
• DNA pTet-GFP from midiprep

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 one of the 96well plates into the 25°C water bath and the other in the 37°C incubator.
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, two times:
   
   1. First prepare a complete amino acid mixture for the extract solution: Add the 25µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 50µl. Each amino acid minus mixture is missing one type of amino acid.
   2. Take an eppendorf tube and add the 50µl of the E.coli complete amino acid mixture.
   3. Add 200µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
   4. Then add 150µl of S30 Extract Circular too.
   5. The final volume of cell extract is: 400µl
5. Each cell extract will be used to test one of the constructs. Label the tubes, identifying which construct it will be used for.
6. Incubate cell extract mixture for CBD in the 37°C incubator.
7. Prepare the different DNA concentrations for pTet construct(concentration of pTet DNA = 500ng/µl):
   1. Concentration 1 = 1µg: Add 4µl of DNA in 36µl nuclease free water.
   2. Concentration 2 = 2µg: Add 8µl of DNA in 32µl nuclease free water.
   3. Concentration 3 = 4µg: Add 12µl of DNA in 28µl nuclease free water.
   4. Concentration 4 = 6µg: Add 16µl of DNA in 16µl nuclease free water.
8. This will give a total volume of 40µl of each DNA concentration. Put each DNA into a seperate, labeled eppendorf tube and place them in the 37°C incubator.

Loading Plate

1. Take the plate out of the incubation.
   1. Follow the schematic for the plate 2 (37°C incubator) and begin by loading 40µl of the in vitro expression system into the right wells.
   2. Tap down the top of the plate to bring down any solution to bottom of the well.
   3. Then add 20µl of purified DNA sample to each well, as indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
   4. Add 20µl of nuclease free water into the two negative control wells, as shown in the schematics.
2. Put 60µl of water into some empty wells in the middle of each plate. These will be used to check for evaporation.
3. 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.
4. Create a file with name referring to the temperature of the plate, under: D:\IGEM\INSERT DATE\CBD\ OTR , for CBD construct. The data from the fluoreometer will be exported here.
5. Each file with the reading should be named as the following:
   • construct-temp-time-date
6. Place the plate in the fluorometer to measure its initial fluorescent reading.
7. After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
8. Start on the next plate, and repeat procedures 2-6.
9. Place the plate in the 37oC incubator.
10. Before placing them in the incubator, wrap aluminium foil around them to prevent photobleaching.
11. Measure the temperature every 30 minutes for each temperature, for 6 hours.

Schematic

Plate 1

Well	Test Construct	Concentration of DNA	In vitro chassis
E5	Nuclease Free Water (Negative control)	0µg	Commercial E.coli extract
E7	Nuclease Free Water (Negative control)	0µg	Commercial E.coli extract
C3	pTet-GFP	1µg	Commercial E.coli extract
C5	pTet-GFP	1µg	Commercial E.coli extract
C7	pTet-GFP (positive control)	2µg	Commercial E.coli extract
C9	pTet-GFP (positive control)	2µg	Commercial E.coli extract
D4	pTet-GFP	4µg	Commercial E.coli extract
D6	pTet-GFP	4µg	Commercial E.coli extract
D8	pTet-GFP	6µg	Commercial E.coli extract
D10	pTet-GFP	6µg	Commercial E.coli extract

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