This method can help answer key questions in the analytical chemistry, biochemistry and biotechnology fields. Such as, what are the changes that occur in the caffeine biosynthesis. The main advantage of this technique, is that it can be applied to invitro plant models that produces caffeine.
To begin, add 10 milliliters of acetone to the lyophilized cells, and seal the flask before mixing with the vortex mixer for 30 seconds. Then, mix the sample at 100 rpm with a rotator for five hours at room temperature. After this, re-suspend the sample with 25 microliters of acetone.
First, apply one microliter of the previously prepared caffeine extract to the silica gel plates. Then, develop the TLC in the chromatography chamber with 10 milliliters of the mobile phase, cyclohexane acetone. Visualize the caffeine bands in short wavelength ultraviolet, with a compact UV lamp.
After this, quantify caffeine levels by densitometry at 273 nanometers. Using filter paper, vacuum filter the previously prepared cell suspension. Then, use a porcelain mortar to macerate the cell sample, with liquid nitrogen and RNA isolation reagent, until it is homogenized.
Next, transfer 500 microliters of the sample to a sterile micro centrifuge tube. Then, add 300 microliters of chloroform isoamyl alcohol, and 300 microliters of equilibrated phenol. Mix the sample with a vortex mixer and centrifuge it at 20, 000G for 15 minutes at four degrees Celsius.
After this, transfer 300 microliters of the upper phase to a clean micro centrifuge tube. Then, add 200 microliters of isopropanol and incubate the tube for one hour at negative 20 degrees Celsius. Add one milliliter of ethanol to the pellet.
Then, centrifuge the tube at 12, 000G for 10 minutes and decant the liquid phase. Next, dry the sample for 1.5 hours at room temperature. Then, re-suspend the RNA extract with 25 microliters of DEPC-treated water.
First, add two micrograms of total RNA extract to a sterile micro centrifuge tube. Then, add one microliter of 10X reaction buffer and one microliter of DNase. Bring the final volume of the reaction to 10 microliters with DEPC-treated water.
Then, mix the sample and centrifuge at 2, 000G for one minute. Next, incubate the sample at 37 degrees Celsius for 30 minutes. After this, add one microliter of 50 micromolar disodium ethylene diamine tetraacetate, to stop the reaction.
Incubate the sample at 65 degrees Celsius for 10 minutes. Then, apply 100 nanograms of RNA to an agarose gel and run the gel. Visualize the integrity of the RNA using a gel photo documentation system.
First, add 2.5 micrograms of total RNA to a micro centrifuge tube. Then, add one microliter of oligo deoxythymidine primer and fill the tube to a final volume of 13 microliters with nuclease-free water. Mix the sample and centrifuge it at 2, 000G for one minute.
Incubate the sample at 65 degrees Celsius for five minutes. And then at four degrees Celsius for two minutes. Next, add four microliters of 5X reaction buffer, two microliters of dNTP mix and one microliter of reverse transcriptase to the sample.
Then, mix the sample gently and centrifuge at 2, 000G for one minute. After this, incubate the sample at 45 degrees Celsius for 50 minutes, and then at 70 degrees Celsius for 10 minutes. Add 7.5 microliters of 2X Taq DNA polymerase and 0.1 micromoles of RAX, to a PCR micro centrifuge tube.
Then, add 0.75 microliters each of forward and reverse primer to amplify the CCS1 gene. After this, add 600 nanograms of CDNA template. Preform the amplification reaction in real time PCR as described in the text protocol.
Then, analyze the data with the PCR software. Weigh out one gram of cellular material and pack it in aluminum foil. After macerating the sample, transfer it to a glass vial and add 2.5 milliliters of extraction buffer.
Next, centrifuge the sample at 20, 000G for 20 minutes at four degrees Celsius. Transfer 500 microliters aliquots into cryogenic vials. With a spectrophotometer, measure the protein concentration of the sample at 562 nanometers using a bicinchoninic acid assay, with bovine seric albumin as the standard.
First, prepare the reaction mixture in a micro centrifuge tube, according to the text protocol. Then, increase the total volume to 200 microliters with 100 millimolar tris HCL. Remember, it is imperative for your own safety to follow proper precautions during preparation of reaction mixture with radioactive material.
Keep the micro centrifuge tube on ice and add a volume of the soluble fraction, containing seven to nine milligrams of protein. Then, vortex the mixture and incubate at 30 degrees Celsius for 30 minutes. After this, centrifuge the samples at 11, 000G for five minutes.
Then, carefully recover a volume of 900 microliters, and transfer the samples to a scintillation vial. Next, evaporate the chloroform to complete dryness in the hood at room temperature. Add five milliliters of scintillation fluid to the vial.
Finally, analyze the radioactivity incorporated into the caffeine using a scintillation counter. In this protocol, the pattern of absorbance for caffeine was analyzed via TLC densitometry, through the visible light spectrum, and reading in maximum absorption at 273 nanometers. The separation curve of caffeine on the TLC plate, showed an RF between 0.34, and 0.39.
The RNA derived from suspended cells exhibits clear separations of the 28s, 18s and 5s RRNA subunits, indicating that the samples were of high quality. Finally, a melt curve was obtained from the QPCR analysis, showing a single amplification product for the caffeine synthase gene. After its development, this technique paved the way for researchers in the fields of biotechnology and biochemistry to explore secondary metabolizing tissue culture from coffee, tea and coco.
Don't forget that working with radioactivity and molecular pathology can be extremely hazardous, and precautions such as the use of gloves, goggles and special equipment for radioactive material shall always be taken when performing this procedure.
