![]() ![]() ![]() For the purpose of this activity, the oxidized form of DCPIP (2,6- dichlorophenol indophenol) will accept two electrons per molecule from photosystem II. In vitro investigation of the process is possible if an electron acceptor is present. There are two photosystems in the chloroplast thylakoid membrane, and light is what drives electron transport through them. The goal here is to rule out the possibility of a reduction in absorbance brought on by the lowering of DCPIP brought about by electron donors that are not involved in photosynthesis. This is because DCPIP can also be reduced by high-energy electrons. Since DCPIP can also be reduced by high-energy electrons coming from other sources, it is necessary for the calculations that give a measure of photosynthetic activity to be based on the changes in absorbance that occur between the samples that are illuminated and those that are kept in the dark. It does this by taking electrons and hydrogen ions from plastoquinone, which is one of the components of the chloroplastic electron transfer chain, which is located within the thylakoid membranes of chloroplasts. DCPIP, in either of its forms, is hydrophobic and has the ability to insinuate itself into cellular membranes. Stop the reaction by heating the probe DNA at 75☌.ġ.Mix equal amount of labelled reference and test DNA in a 1.5mL microcentrifuge tube approximately.Ģ.Add 5mL Human Cot-1 DNA and make up to 100mL with dH2O.ģ.Add 10mL 3M sodium acetate solution into the DNA sampleĤ.Add 250mL cold absolute ethanol to the mixture and mix well.ĥ.Incubate the mixture at –20☌ for at least 30-60 minutes.Ħ.Centrifuge the mixture at 14000rpm at 4☌ for 20 minutes.ħ.Discard the supernatant and vacuum dry the DNA probe.Ĩ.Store the DNA probe at –20☌ until use.An indication of redox reactions, DCPIP is reduced by the addition of two electrons and two hydrogen ions to each of its molecules. It is recommended that just prepare the slide when you use it as the slides is not preferred for hybridization if incubate for more than 2 weeks.ĭNA probe labelling (Nick Translation labelling):ġAdd the following components to the tube:ģ.Incubate at 15☌ for 2 hours and then temporarily stop the reaction by placing the tube at -20☌.Ĥ.Check the size of the labeled probes by gel electrophoresis, looking for the peak size between 300-800 bp DNA fragments.ĥ.If the desired probe size is reached, goto step 6 otherwise, continued the incubation at step 4 until adequate size of DNA is obtained.Ħ. Add 8mL fixative to the remaining and store it at -20☌ until use.ġ1.Incubate the slides at 37☌ for 3 days before use. After the third rinse and spin, leave enough supernatant to make a milky Cell suspension and prepare the normal metaphase slide now. Wash the pellet with fresh fixative twice. Incubate at -20☌ for overnight.ġ0.Centrifuge at 1000 rpm for 5 minutes. Add the Cell s to the cold fixative in drop by drop fashion, and mix them well gently. Add approximately 5mL of cold fixative to the tube. Pipette up all the material with glass pasteur pipette. ![]() Be-care not to disrupt the underneath pellet.ĩ.Resuspend the pellet thoroughly. Discard the supernatant and leave 0.5mL above the pellet. Meanwhile, prepare fresh fixative (1X 100% glacial acetic acid, 3X 100% methanol) and place on ice bath to chill until use.Ĩ.Centrifuge at 1000 rpm for 5 minutes at room temperature. Make up to 14mL solution with prewarmed 0.075M KCl and place in 37☌ water bath for 15-20 minutes (depending on the species and mitotic inhibitors used). Resuspend the pellet well.ħ.Add 2mL prewarmed (37☌) 0.075M KCl drop by drop and mix the tube gently. Discard the supernatant and leaving 0.5mL on top of the pellet. Centrifuge at 1000 rpm for 5 minutes at room temperature. If CO2 incubator is unavailable, use a closed system by capping the tubes tightly.ĥ.Add 50mL 10mg/mL colcemid for 45 minutes at 37☌ on the last day of culture.Ħ.Transfer all the content to a 15-mL centrifuge tube. Centrifuge at 14000 rpm for 20mins at room temperature.Ģ.Equally transfer the first and the second layers to three T25 culture flasks in a total volume of 10mL completed medium with 300mL ACS and 100mL PHA.ģ.Incubate in the humidified incubator with 5% CO2 at 37☌ for 3 days. 1.Collect 5mL whole blood from volunteer and transfer to a centrifuge tube with 500mL ACS. ![]()
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