Overview of our Research Protocol

 

P.s. A few hours ago, I noticed that my account had uploaded some strange Lab notes and comments. It turned out to be due to a series of minor hacks intended as pranks, and the culprits were caught. Again, I apologize for those posts. After that, I've changed my password. T

1. Objectives

The objectives of our research is to figure out the relationships between presence of Inverted Repeat inside of chloroplast DNA of certain plant(for us, 'certain' plants are Senegalia senegal and its 5 sister group species) and presence of symbiotic bacteria associated with the certain plant and wheather the certain plant is deciduous(; it meant the plant drops leaves under certain condition alike temperature drop, lower humidity,....etc.). For this, we need to analysis gene sequences of those plant, including their chloroplast DNA sequences.

2. Procedures

Most of our researches is processed inside of laboratory. Below is some important procedures for this research. Take a look please.(More detailed update is scheduled to the near future. Stay tuned!)

• Sampling target plants' specimens

Meaning of Sampling: In biological experiment, it is not possible to collect complete information about a population. If the number of pods/plant is to be collected from a field then it is really time consuming and also rarely possible to do it. Then few plants are taken into account for studying the whole population of plant in that field. The method by which only few items are selected from the population in such a way so that they will represent the population in unbiased way is called sampling. The size of sample is an important factor in statistical analysis which depends on the number of sampling units selected from a population for investigation. The size should not be too big or too small. Taking only 10 values from a plot of 1000 plants will give erroneous result and also it is difficult to handle with 1000 number of values. And in microscopy, sampling can also be the process of selecting certain cells, organelles, etc from the target organism.

Why Sampling is Essential? A. Sampling saves time, the data can be collected and summarised more quickly with a sample than a complete count of the whole population. B. In case of infinite population, sampling is the only method for statistical analy­sis. C. Sampling reduces the cost of experiment because only a few selected items are studied in sampling.

 

For analyzing specific genes, we need to do sampling work first.

• Extract their genes and run sequencing, by using sequencing machine, PCR and Electrophoresis,....,etc.(We'll disclose detailed methods by pictures and videos, as soon as this project starts in 2023 or 2025. Stay tuned!) Here is the known protocols below.

Step 1. Breaking cells open to release the DNA The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing, and put into a solution containing salt. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA. A detergent is then added. The detergent breaks down the lipids in the cell membrane and nuclei. DNA is released as these membranes are disrupted. Step 2. Separating DNA from proteins and other cellular debris To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as possible. This can be done by a variety of methods. Often a protease ( protein enzyme) is added to degrade DNA-associated proteins and other cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the sample. Step 3. Precipitating the DNA with an alcohol Finally, ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. By gently stirring the alcohol layer with a sterile pipette, a precipitate becomes visible and can be spooled out. If there is lots of DNA, you may see a stringy, white precipitate. Step 4. Cleaning the DNA The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer and ready to use. Step 5. Confirming the presence and quality of the DNA For further lab work, it is important to know the concentration and quality of the DNA. Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can be used to show the presence of DNA in your sample and give an indication of its quality.

• Extract chloroplasts from sampled plants by using centrifugation method.

 

After centrifugation, we need to leave intact chloroplasts.

• Isolate the target gene fragments of the sampled gene by using PCR & Electrophoresis techniques and specific primers and gene-editing agents(e.g. CRISPR).

 

Overall protocols including PCR and Electrophoresis.

• Detailed analysis of functional and sequence associations between sequenced genomes.

• Perform controlled experiment with the plants. Independent Variable is the presence of Inverted Repeat sequences(We'll have to use gene editing tools to erase or add Inverted Repeat sequences inside of those plants' chloroplast DNA.)

3. Analysis

We are planning to analyze extracted genes by using some instruments like Promega Maxwell® Instruments or Applied Biosystems™ 3730xl DNA Analyzer.

More detailed analysis method will be updated as soon as possible. Thank you for waiting :)

4. References